A multiplex recombinase polymerase amplification assay combined with CRISPR/Cas12a for the detection of respiratory syncytial virus and respiratory adenovirus.

OBJECTIVE: Respiratory syncytial virus (RSV) and respiratory adenovirus (ADV) are two common pathogens that cause acute respiratory tract infections in children. We aimed to develop a rapid method for detecting both pathogens simultaneously. METHODS: The recombinase polymerase isothermal amplification (RPA) method was combined with the CRISPR/Cas detection system. The assay's specificity and sensitivity were explored by designing RPA primers and CRISPR RNAs (crRNAs) through multi-sequence comparisons, optimizing the reaction conditions, and using a fluorescent reading device. The consistency of the test results of 160 clinical pharyngeal swab samples was studied using quantitative polymerase chain reaction (qPCR) results as a comparative control. RESULTS: RSV and ADV could be detected at levels as low as 104 copies/mL and 103 copies/mL, respectively, within 50 minutes with no cross-reactivity with other similar pathogens. For the clinical samples, compared with the qPCR method, the sensitivities for RSV and ADV were 98.1% and 91.4%, respectively, and the detection specificities were both 100%. The Kappa values were greater than 0.95, suggesting a high degree of consistency. CONCLUSION: This method for detecting RSV and ADV is rapid, sensitive, and specific. It can accurately detect mixed infections in a timely manner, making it suitable for use in areas with scarce healthcare resources.

Epidemiological characteristics, genotypes, genetic relationships and macrolide resistance of 31 Bor-detella pertussis strains isolated from infants and young children in Shenzhen / 中华微生物学和免疫学杂志

Objective To understand the epidemiological characteristics, genomic variations and macrolide resistance of Bordetella pertussis ( B. pertussis) strains circulating in Shenzhen with clinical data analysis, genotype profiling, phylogenetic analysis and antimicrobial susceptibility test. Methods Clinical data of patients with pertussis in Shenzhen Children's Hospital were collected from the electronic medical re-cord system. Genome sequences of 31 B. pertussis isolates were analyzed with next-generation sequencing and de novo assembled. Multilocus sequence typing (MLST) was performed to identify their sequences types. Sequence alignment by BLASTn was used to identify virulence genotypes and mutations in 23S rRNA gene. A phylogenetic tree was constructed to analyze the relationships among them. E-test was used to identify ma-crolide resistance. Results All of the 31 B. pertussis strains were identified as sequence type-2 (ST-2) by MLST with diverse virulence genotypes. Two were prn-deficient strains. Based on the phylogenetic tree, all of the isolates were distant from vaccine strains. Nineteen isolates were resistant to erythromycin with A2047G mutation in 23S rRNA. Conclusions The virulence genotypes of B. pertussis strains in Shenzhen were diverse with increasing non-vaccine genotypes. Macrolide-resistant strains were prevalent. This study might provide reference for improving the prevention, management and vaccination strategy of pertussis.