Real-Time Reverse Transcription–Polymerase Chain Reaction Assay for SARS-associated Coronavirus

Emery, Shannon; Erdman, Dean D.; Bowen, Michael D.; Newton, Bruce R.; Winchell, Jonas M.; Meyer, Richard F.; Tong, Suxiang; Cook, Byron T.; Holloway, Brian P.; McCaustland, Karen A.; Rota, Paul A.; Bankamp, Bettina; Lowe, Luis; Ksiazek, Thomas G.; Bellini, William J.; Anderson, Larry J.

doi:10.3201/eid1002.030759

Cited by 315 publications

(266 citation statements)

References 14 publications

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“…The triplicate assay, in which a sample is analyzed 3 times, is the generally accepted method used in qPCR analysis, be it chytridiomycosis (Boyle et al 2004), biomedical (Donovan et al 2000, Ginzinger 2002, Emery et al 2004), or microbiological (Helps et al 2001, Okano et al 2004 research. Triplicate assays are thought to reduce the chances of both false positives (i.e.…”

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confidence: 99%

Cost efficiency in the detection of chytridiomycosis using PCR assay

Kriger¹,

Hero²,

2006

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Section: Resale or Republication Not Permitted Without Written Consenmentioning

confidence: 99%

Cost efficiency in the detection of chytridiomycosis using PCR assay

Kriger¹,

Hero²,

2006

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“…The thermal cycling was carried out with an AB Prism 7900HT thermal cycler with 48°C for 30 min, 95°C for 5 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. The presence of intact RNA in the samples was confirmed with primers specific for RNase P RNA (8). Four positive controls containing known copy numbers of transcribed RV RNA were run on each plate as quantification standards.…”

Section: Methodsmentioning

confidence: 99%

Confirmation of Rubella within 4 Days of Rash Onset: Comparison of Rubella Virus RNA Detection in Oral Fluid with Immunoglobulin M Detection in Serum or Oral Fluid

et al. 2009

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Rubella virus infection is typically diagnosed by the identification of rubella virus-specific immunoglobulinSymptomatic rubella is characterized by a mild fever and a maculopapular rash of short duration. The clinical diagnosis of rubella is unreliable, and many rash illnesses, such as those caused by measles virus and parvovirus B19, mimic rubella (2). Therefore, laboratory confirmation is essential for the diagnosis of rubella and is typically done by testing serum samples for rubella virus (RV)-specific immunoglobulin M (IgM) antibodies. Serum IgM and IgG responses to RV develop rapidly in the first few days after the onset of rash. However, approximately 50% of samples collected on the day of rash onset test negative for RV-specific IgM antibodies (1, 9, 17). Often, only a single serum sample taken near the time of rash onset is available, resulting in the lack of serologic confirmation of many rubella cases. Thus, the development of a rapid laboratory diagnostic tool for the confirmation of rubella within the first few days of symptom onset would improve the ability to confirm rubella.The isolation of virus in cell culture or the detection of viral RNA by reverse transcription-PCR (RT-PCR) also provides reliable evidence of RV infection (26). Unfortunately, blood is not a good sample for use for the detection of RV, because the highest viral titers in blood typically occur before the onset of rash and virus is undetectable in blood by 2 days after rash onset (6). The virus titer in throat swabs, however, usually reaches a peak titer on the day of rash onset and the titers in throat swabs decline more slowly than those in blood, so that virus can be detected for up to 5 to 7 days after rash onset (6). Several RT-PCR assays for the detection of the RV genome in clinical samples have been described (3,7,15,16,20,25). Templates for the determination of viral sequences for molecular epidemiology can also be made by using RT-PCR.The use of alternative specimens could help reduce the obstacles to specimen collection, storage, and transport in the field (22). Oral fluid (OF), which is collected by rubbing an absorptive device between the gum and the cheek, can be obtained by a method that is relatively noninvasive, is easier to obtain than blood, and has the advantage that it can be used for both RVspecific antibody detection and RV genome detection (12,19,20). Currently, in the United Kingdom, OF samples from notified clinically diagnosed cases are collected between 1 and 6 weeks after the onset of symptoms and are transported by mail to the Central Public Health Laboratory, where they are tested for specific antibody and viral RNA by RT-PCR. By the use of this strategy, specimens from 54.6% of rubella notifications from 1995 through 2001 were obtained for laboratory testing and specimens from 12.7% of the rubella notifications were confirmed to represent rubella cases (20,21).

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“…Each run included template and nontemplate controls. Specimen extracts also were tested for the human RNase P gene to monitor specimen quality as previously described (10).…”

Section: Methodsmentioning

confidence: 99%

Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses

Lu¹,

Holloway

Dare³

et al. 2008

J Clin Microbiol

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266

229

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Human rhinoviruses (HRVs) are important contributors to respiratory disease, but their healthcare burden remains unclear, primarily because of the lack of sensitive, accurate, and convenient means of determining their causal role. To address this, we developed and clinically validated the sensitivity and specificity of a real-time reverse transcription-PCR (RT-PCR) assay targeting the viral 5 noncoding region defined by sequences obtained from all 100 currently recognized HRV prototype strains and 85 recently circulating field isolates. The assay successfully amplified all HRVs tested and could reproducibly detect 50 HRV RNA transcript copies, with a dynamic range of over 7 logs. In contrast, a quantified RNA transcript of human enterovirus 68 (HEV68) that showed the greatest sequence homology to the HRV primers and probe set was not detected below a concentration of 5 ؋ 10 5 copies per reaction. Nucleic acid extracts of 111 coded respiratory specimens that were culture positive for HRV or HEV were tested with the HRV real-time RT-PCR assay and by two independent laboratories that used different in-house HRV/HEV RT-PCR assays. Eighty-seven HRVculture-positive specimens were correctly identified by the real-time RT-PCR assay, and 4 of the 24 HEVpositive samples were positive for HRV. HRV-specific sequences subsequently were identified in these four specimens, suggesting HRV/HEV coinfection in these patients. The assay was successfully applied in an investigation of a coincidental outbreak of HRV respiratory illness among laboratory staff.

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Real-Time Reverse Transcription–Polymerase Chain Reaction Assay for SARS-associated Coronavirus

Cited by 315 publications

References 14 publications

Cost efficiency in the detection of chytridiomycosis using PCR assay

Cost efficiency in the detection of chytridiomycosis using PCR assay

Confirmation of Rubella within 4 Days of Rash Onset: Comparison of Rubella Virus RNA Detection in Oral Fluid with Immunoglobulin M Detection in Serum or Oral Fluid

Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses

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