Objective:To develop a single-tube one-step multiplex nested real-time reverse transcription polymerase chain reaction (RT-PCR) assay for the simultaneous detection of 2019-nCoV, influenza A virus, influenza B virus and internal-control with human-derived gene.Methods:This study included 30 positive specimens for 2019-nCoV nucleic acid detection and 336 screening specimens collected from the arrivals at Guangzhou Baiyun Airport between February 2020 and February 2022. Sixty-four positive specimens of other respiratory pathogens were also collected from the arrivals at Guangzhou Baiyun Airport during the three-year period before the occurrence of COVID19 outbreak in 2020, and 7 positive viral strains of respiratory pathogens were provided by collaborative laboratories. In order to establish a set of multiplex nested real-time RT-PCR assay, a group of primers and probe combinations for a multiplex nested real-time RT-PCR was designed and screened according to a selection of nucleotide conserved regions of the ORF and N genes of 2019-nCoV and the M gene of influenza A and B viruses, while nested amplification primers and probe for the internal-control with human-derived gene were introduced. Then the prepared pseudovirus-positive quality control and sample discs were applied to evaluate the sensitivity and specificity. Clinical specimens were performed to validate the applicability of the method.Results:The results show that the established one-step multiplex nested real-time RT-PCR assay can specifically detect 2019-nCoV and influenza A and B viruses, with the limit-of-detection of about 125 copies/ml for 2019-nCoV and about 250 copies/ml for influenza A and B viruses. Totally 101 positive samples of various respiratory pathogens were detected, showing that the detection sensitivities of 2019-nCoV and influenza A and B viruses were 96.67%, 92.86% and 96.15%, respectively, with the specificity of 100%. No false-positive detection was found in the applied detection of more than 300 clinical samples.Conclusions:A one-step multiplex nested real-time RT-PCR assay for 2019-nCoV, influenza A and B viruses and human-derived gene internal-control was developed. The assay has good sensitivity and specificity and can be used for rapid screening of 2019-nCoV and influenza A and B viruses in high-volume samples.

Objective To investigate the prevalence of Anisakis infections in coastal marine fishes and awareness of anisakiasis control knowledge among local residents in Yantai City, Shandong Province in 2021, so as to provide insights into formulation of anisakiasis control interventions. Methods Marine fishes were purchased from Shunxin Port, Yantai City, Shandong Province in November 2021, and the presence of Anisakis was detected in different species of fishes and different fish sites. The correlations between body length and weight of marine fish and intensity of Anisakis infections were examined using Spearman’s rank correlation analysis, and the dietary habits and anisakiasis control knowledge were investigated using questionnaire surveys among local residents. Results A total of 201 marine fishes belonging to 20 species were dissected, and Anisakis was detected in 77 marine fishes (38.31%) belonging to 11 species (55.00%), with a mean infection intensity of 45.04 parasites per fish (3 468/77). Spearman’s rank correlation analysis revealed that the body length (r_s = 0.74, P < 0.05) and weight (r_s = 0.79, P < 0.01) of the monkfish correlated positively with the intensity of Anisakis infections, and the body length (r_s = 0.68, P < 0.05) of the flatfish correlated positively with the intensity of Anisakis infections, while no correlations were examined between the body length or weight of other marine fishes and the intensity of Anisakis infections. Of all respondents, 53.38% men and 56.67% women did not know anisakiasis control knowledge at all, and there was a significant difference in the proportion of respondents using separate chopping boards for raw and cooked food from different villages (χ² = 17.89, P < 0.01), while there was an age-specific proportion of respondents with habitats of eating raw or semi-raw seafood (χ² = 28.27, P < 0.01). Conclusions The prevalence and intensity of Anisakis infections were high in coastal marine fishes in Yantai City in 2021, and the awareness of anisakiasis control knowledge was low among local residents. Intensified health education pertaining to anisakiasis control knowledge is recommended to reduce the risk of Anisakis infections.

The lung is one of the most common sites for cancer metastasis. Collagens in the lung provide a permissive microenvironment that supports the colonization and outgrowth of disseminated tumor cells. Therefore, down-regulating the production of collagens may contribute to the inhibition of lung metastasis. It has been suggested that miR-29 exhibits effective anti-fibrotic activity by negatively regulating the expression of collagens. Indeed, our clinical lung tumor data shows that miR-29a-3p expression negatively correlates with collagen I expression in lung tumors and positively correlates with patients' outcomes. However, suitable carriers need to be selected to deliver this therapeutic miRNA to the lungs. In this study, we found that the chemotherapy drug cisplatin facilitated miR-29a-3p accumulation in the exosomes of lung tumor cells, and this type of exosomes exhibited a specific lung-targeting effect and promising collagen down-regulation. To scale up the preparation and simplify the delivery system, we designed a lung-targeting liposomal nanovesicle (by adjusting the molar ratio of DOTAP/cholesterol-miRNAs to 4:1) to carry miR-29a-3p and mimic the exosomes. This liposomal nanovesicle delivery system significantly down-regulated collagen I secretion by lung fibroblasts in vivo, thus alleviating the establishment of a pro-metastatic environment for circulating lung tumor cells.