An Ultra-Rapid Real-Time RT-PCR Method Using the PCR1100 to Detect Severe Acute Respiratory Syndrome Coronavirus-2.

The disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Wuhan, China, in December 2019, has rapidly spread worldwide. SARS-CoV-2 is usually detected via real-time reverse-transcription polymerase chain reaction (RT-PCR). However, the increase in specimen load in institutions/hospitals necessitates a simpler detection system. Here, we present an ultra-rapid, real-time RT-PCR assay for SARS-CoV-2 detection using PCR1100 device. Although PCR1100 tests only one specimen at a time, the amplification period is less than 20 min and the sensitivity and specificity match those of conventional real-time RT-PCR performed on large instruments. The method is potentially helpful when daily multiple SARS-CoV-2 testing is needed, for example to confirm virus-free status prior to patient discharge.

An Ultra-Rapid Real-Time RT-PCR Method Using PCR1100 for Detecting Human Orthopneumovirus.

Human orthopneumovirus, also known as the respiratory syncytial virus (RSV), is a leading cause of respiratory tract infections in children worldwide. The World Health Organization has taken steps toward establishing a global surveillance system for RSV, based on the global influenza surveillance and response system initiated in 2015. The US Centers for Disease Control and Prevention (CDC) has developed a genetic detection method based on real-time reverse transcription polymerase chain reaction (RT-PCR), which is used in global RSV surveillance. In Japan, immunoassay-based rapid antigen detection kits are widely used for the detection of RSV. In this study, an ultra-rapid real-time RT-PCR method for the rapid detection of RSV was developed using the PCR1100 device based on the US CDC assay in order to detect RSV in comparable time to rapid test kits. The ultra-rapid real-time RT-PCR could detect RSV viral RNA in less than 20 min while maintaining sensitivity and specificity comparable to conventional real-time RT-PCR using large installed instruments. Furthermore, combining ultra-rapid real-time RT-PCR with the M1 Sample Prep kit reduced the total working time for the detection of RSV from clinical specimen to less than 25 min, suggesting this method could be used for point-of-care RSV testing.

Ultra-Rapid Real-Time RT-PCR Method for Detecting Middle East Respiratory Syndrome Coronavirus Using a Mobile PCR Device, PCR1100.

Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) is usually diagnosed through highly sensitive and specific genetic tests such as real-time reverse transcription polymerase chain reaction (RT-PCR). Currently, two real-time RT-PCR assays targeting the upE and ORF1a regions of the MERS-CoV genome are widely used, and these are the standard assays recommended by the World Health Organization (WHO). The MERS outbreaks to date suggest that rapid diagnosis and subsequent isolation of infected patients, particularly superspreaders, are critical for containment. However, conventional real-time RT-PCR assays require large laboratory instruments, and amplification takes approximately 2 h. These disadvantages limit rapid diagnosis. Here, an ultra-rapid real-time RT-PCR test was established comprising a multiplex assay for upE and ORF1a running on a mobile PCR1100 device. As few as five copies of the MERS-CoV RNA can be detected within 20 min using the standard WHO assays in the mobile PCR device, with the sensitivity and specificity being similar to those of a conventional real-time PCR instrument such as the LightCyler, thereby enabling timely intervention to control MERS-CoV infection.