Comparison of 9 different PCR primers for the rapid detection of severe acute respiratory syndrome coronavirus using 2 RNA extraction methods

Abstract

The sensitivity and specificity of various severe acute respiratory syndrome coronavirus (SARS-CoV) PCR primer and probe sets were evaluated through the use of commercial kits and in-house amplification formats. Conventional and real-time PCR assays were performed using a heat-block thermocycler ABI 9600, the Roche LightCycler version 1.2, or the ABI 7000 Sequence Detection System. The sensitivity of all primers was between 0.0004 and 0.04 PFU with viral cell lysate and between 0.004 and 0.4 PFU in spiked stool specimen per PCR assay. The primer sets for real-time PCR assays were at one least 1 log more sensitive than the primer sets used in the conventional PCR. A panel of viruses including swine gastroenteritis virus, bovine coronavirus, avian bronchitis virus (Connecticut strain), avian bronchitis virus (Massachusetts strain), human coronaviruses 229E and OC43, parainfluenza virus (type III), human metapneumovirus, adenovirus, respiratory syncytial virus, and influenza A were tested by all assays. All real-time PCR assays used probe-based detection, and no cross-reactivity was observed. With conventional PCR, analysis was performed using agarose gel electrophoresis and multiple nonspecific bands were observed. Two commercial extraction methods, magnetic particle capture and silica-based procedure were evaluated and the results were comparable. The former was less laborious with shorter time for completion and can easily be adapted to an automated system such as the MagNa Pure-LC, which can extract nucleic acid from clinical samples and load it into the sample capillaries of the LightCycler. As exemplified by this study, the continued refinement and evaluation of PCR procedures will greatly benefit the diagnostic laboratory during an outbreak of SARS.

SYBR Green real-time reverse transcription-polymerase chain reaction assay for the generic detection of coronaviruses

“…The sensitivity of our method is similar to the sensitivity of other real-time PCR assays based on the ORF1b for the specific detection of SARS-CoV, including the commercial SARS-CoV Quantification kit (Roche, Penzberg, Germany) [6,7,19,22,25,27]. The RealArt HPA coronavirus LC kit (Artus, Hamburg, Germany) nevertheless seems to be more sensitive, with a detection level down to 0.5-1.5 SARS-CoV RNA copy per reaction [4]. Targeting the N gene has been postulated to be a better choice to improve the sensitivity of the PCR as this gene is supposed to have the most abundant copy number during viral replication [35].…”

An Outbreak of Human Coronavirus OC43 Infection and Serological Cross‐Reactivity with SARS Coronavirus

“…The NML has maintained rigorous quality control since then, with no evidence of similar findings. Since 2003, there has been some effort to refine RT-PCR testing for SARS-CoV generally, and most experts now caution against the use of nested methods for routine diagnostic testing (35). The present investigation underscores the fact that laboratory testing is but one way to form inferences on the etiology of outbreaks, and cannot replace scrupulous clinical and epidemiological observation.…”

Paramagnetic particles coupled with an automated flow injection analysis as a tool for influenza viral protein detection

“…Similar approaches have implemented microfluidic platforms that can detect pathogenic avian influenza virus H5N1 in a throat swab sample. The technique coupled with polymerase chain reaction uses magnetic forces to manipulate a free droplet containing superparamagnetic particles . These approaches are so sensitive, as Mizutani et al showed, that they were able to isolate five particles from the influenza virus .…”