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Objective@#To establish a real-time fluorescence recombinase acid amplification (RAA) method for the detection of adenovirus type 3(HAdV-3)without extraction nucleic acid.@*Methods@#According to the conserved sequence of adenovirus type 3 gene, a pair of primers and a probe were designed, and a real-time fluorescence RAA without extracting nucleic acid was established and optimizing the condition of DNA-free extraction. The sensitivity of the method was analyzed by a series of dilution and the specificity of the method was evaluated by detecting the original samples of other respiratory viruses. The clinical samples of HAdV-3 were detected and compared with the traditional real-time fluorescence quantitative PCR method for nucleic acid extraction.@*Results@#The sensitivity of the real-time fluorescence RAA method was as high as that of qPCR in the detection of 10 series diluted HAdV-3 strains. The highest corresponding CT value of qPCR was 36.87. The sensitivity of the real-time fluorescence RAA method was similar to that of the real-time fluorescence quantitative PCR method . There was no cross-reaction to other common types of respiratory viruses. The two method were used to detect 56 clinical samples at the same time, and the result were completely consistent.@*Conclusions@#We provide the first report of the real-time fluorescent RAA assays for the detection of HAdV-3 without extracting nucleic acid and it has high sensitivity and specificity. Is suitable for rapid detection of HAdV-3 in clinical laboratories and on-site unite.
RESUMO
Objective@#To compare the detection rate of herpes virus and enterovirus (EV) in paired cerebrospinal fluid and serum samples of patients with viral encephalitis.@*Methods@#A total of 109 paired cerebrospinal fluid and serum specimens were collected from patients who were clinically diagnosed with suspected viral meningitis in Children′s Hospital of Hunan from December 2017 to February 2018. One-step nested real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) and real-time fluorescence quantitative PCR were used to detect enterovirus and herpes virus respectively and the detection rates of different virus and sample types were analyzed. SPSS 17.0 was used for statistical analysis of the test result .@*Results@#Among the 109 pairs of specimens, the positive rates of human herpes virus type 6 (HHV6), herpes simplex virus-1 (HSV1), Epstein-Barr virus (EBV), cytomegalovirus (CMV) and enterovirus group A type 71(EV-A71) in serum were 7.34%, 4.59%, 7.34%, 9.17% and 10.09%, respectively, and in cerebrospinal fluid were 5.50%, 2.75%, 0, 5.50%, and 6.42%, respectively. The result showed that there were statistically significant differences between the two types of specimens for herpes virus and enterovirus (P<0.05). In cerebrospinal fluid and serum samples, the longest time for EV-A71 positive detection was 2 and 7 days after onset, respectively; the longest time for CMV positive detection was 3 and 26 days after onset, respectively; the longest time for HHV6 positive detection was 7 and 8 days after onset, respectively; the longest time for HSV1 positive detection was both 12 days after the onset; in serum samples, the longest time for EBV positive detection was10 days after onset, but in cerebrospinal fluid, no EBV was detected within 10 days of onset.@*Conclusions@#EV-A71 is the most prevalent pathogen causing viral encephalitis in hunan, the overall positive rate of virus in serum samples was higher than that in cerebrospinal fluid samples. Virus stays longer in serum than in cerebrospinal fluid. It is suggested that the time is of great significance for the pathogen detection of children with viral encephalitis, the specimen type can be selected reasonably according to the time of onset.