Improving the efficiency and biosafety of respiratory syncytial virus identification using a nucleic acid extraction-free reagent.

Respiratory syncytial virus (RSV) is the most important virus that causes lower respiratory tract disease in children; efficient viral identification is an important component of disease prevention and treatment. Here, we developed and evaluated a ready-to-use (RTU) nucleic acid extraction-free direct reagent for identification of RSV (RTU-Direct test) in clinical samples. The limit of detection (LOD) of the RSV RTU-Direct test was consistent with the LOD of the standard test using extracted nucleic acids. The virus inactivation ability of RTU-Direct reagent was confirmed by viral infectivity assays involving RTU-Direct-treated samples containing RSV and human coronavirus OC43. RSV RNA stability was significantly better in RTU-Direct reagent than in conventional virus transport medium (VTM) at room temperature and 4°C (p < 0.05). The clinical performance of the RTU-Direct test was evaluated using 155 respiratory specimens from patients with suspected RSV infection. Positive agreement between the RTU-Direct test and the VTM standard test was 100% (42/42); negative agreement was 99.1% (112/113), and the kappa statistic was 0.968 (p < 0.001). The distributions of Ct values did not significantly differ between the RTU-Direct test and the standard test (p > 0.05). Overall, the RTU-Direct reagent can improve the efficiency and biosafety of RSV detection, while reducing the cost of detection.

Establishment and evaluation of a 30-minute detection method for SARS-CoV-2 nucleic acid using a novel ultra-fast real-time PCR instrument.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is still raging worldwide. Efficient, fast and low-cost severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection methods are urgently needed. METHODS: A rapid PCR temperature change mode was explored by moving the reaction tube between the independent temperature modules with large temperature differences and a portable ultra-fast real-time PCR instrument were developed. We established a rapid SARS-CoV-2 test method using the ultra-fast real-time PCR instrument, a China Food and Drug Administration-certified SARS-CoV-2 reagent and optimized reaction condition. The analytical and clinical performances of the rapid tests were evaluated by comparing with the standard SARS-CoV-2 tests. RESULTS: The new temperature change mode can effectively shorten the amplification reaction time and be successfully used in the development of the ultra-fast real-time PCR instrument. The rapid SARS-CoV-2 test method was established and the time to yield results were greatly shortened from 81 min of the standard test to 31 min. Specificity of the rapid test was assessed and no non-specific amplification (0/63) was observed. The limits of detection of the rapid and standard tests were similar. Clinical performance was evaluated using 184 respiratory specimens from patients with suspected SARS-CoV-2 infection. The positive agreement between the rapid and standard tests was 100% (67/67), the negative agreement was 97.4% (114/117), and the kappa statistic was 0.965 (P<0.001). No significant differences in the Ct values for each target gene were observed between the rapid test and the standard test (P>0.05). CONCLUSIONS: We had developed a 30-minute detection method for SARS-CoV-2 nucleic acid using a novel ultra-fast real-time PCR instrument. The rapid test method may impact on patient management.

Two novel human coronavirus OC43 genotypes circulating in hospitalized children with pneumonia in China.

HCoV-OC43 is one of the mildly pathogenic coronaviruses with high infection rates in common population. Here, 43 HCoV-OC43 related cases with pneumonia were reported, corresponding genomes of HCoV-OC43 were obtained. Phylogenetic analyses based on complete genome, orf1ab and spike genes revealed that two novel genotypes of HCoV-OC43 have emerged in China. Obvious recombinant events also can be detected in the analysis of the evolutionary dynamics of novel HCoV-OC43 genotypes. Estimated divergence time analysis indicated that the two novel genotypes had apparently independent evolutionary routes. Efforts should be conducted for further investigation of genomic diversity and evolution analysis of mildly pathogenic coronaviruses.

Discovery of a subgenotype of human coronavirus NL63 associated with severe lower respiratory tract infection in China, 2018.

Human coronavirus NL63 (HCoV-NL63) is primarily associated with common cold in children, elderly and immunocompromised individuals. Outbreaks caused by HCoV-NL63 are rare. Here we report a cluster of HCoV-NL63 cases with severe lower respiratory tract infection that arose in Guangzhou, China, in 2018. Twenty-three hospitalized children were confirmed to be HCoV-NL63 positive, and most of whom were hospitalized with severe pneumonia or acute bronchitis. Whole genomes of HCoV-NL63 were obtained using next-generation sequencing. Phylogenetic and single amino acid polymorphism analyses showed that this outbreak was associated with two subgenotypes (C3 and B) of HCoV-NL63. Half of patients were identified to be related to a new subgenotype C3. One unique amino acid mutation at I507 L in spike protein receptor binding domain (RBD) was detected, which segregated this subgenotype C3 from other known subgenotypes. Pseudotyped virus bearing the I507 L mutation in RBD showed enhanced entry into host cells as compared to the prototype virus. This study proved that HCoV-NL63 was undergoing continuous mutation and has the potential to cause severe lower respiratory disease in humans.