ABSTRACT
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.
ABSTRACT
Objective To examine the prevalence of chikungunya virus in brain tissue samples from rat?like animals in Xiamen, Shenzhen and Guangzhou, and to explore whether the rat?like animals are potential sources of human chikungunya fever infections and the host of the virus. Methods Rat?like animals were trapped in residential areas, city parks, hospitals, markets and schools in Xiamen, Shenzhen and Guangzhou (Yuexiu and Baiyun districts) between January 2013 and June 2016. Brain tissue samples of the trapped animals were collected under sterile. Chikungunya virus was detected by using reverse transcription polymerase chain reaction (RT?PCR). Results Totally 1092 rat?like animals were trapped, which belonged to 7 species, 3 genera, 2 families, 2 orders. Rattus norvegicus was the dominant species in the indoor environment, Rattus losea was dominant in wild environment, and 1092 brain tissue samples were collected. No detectable chikungunya virus was found in the brain tissue samples by RT?PCR. Conclusion There is a low possibility that rat?like animals act infectious sources of human chikungunya fever infections and the host of the virus.
ABSTRACT
We detected Zika virus (ZIKV) in a febrile case returning from Suriname and entry China from Guangzhou Baiyun International Airport Port.Serum and saliva samples were collected from a suspected case returning from Suriname.We detected ZIKV RNA using real-time fluorescence RT-PCR methods by both in-house reagent and commercial detection kits.RT-PCR detection was carried out with saliva sample and sequence analysis was performed.Phylogenetic tree was constructed to analyze the source of imported cases.Real-time fluorescent RT-PCR result showed that saliva was detected ZIKV RNA positive while for serum was weakly positive.A specific 1 500 bp fragment in size was amplified with saliva sample by RT-PCR.Sequence analysis showed 99％ homologous to the corresponding sequence of Brazil ZIKV (GenBank No.KX197250).Phylogenetic tree indicated it was located on African lineage.According to the epidemiological investigation results,clinical manifestations and nucleic acid detection of case,the suspected case was confirmed to infect Zika virus,being the first case from Suriname into Guangdong Province.