| Part one A triplex quantitative real-time PCR assay for differential detection of human adenovirus serotypes 2,3 and 7Objective: A triplex quantitative real-time PCR assay was established then realized a single tube simultaneous detection of human adenovirus(HAdV)serotypes 2,3,7.It provides a rapid and convenient method for diagnosis of HAdV serotypes 2,3,7.Methods:(1)First,the HAdV complete genome sequence and serotypes 2,3 and 7 Hexon gene sequence were download from GenBank.(2)The universal primers of HAdV were quoted from previous study.Through Vector NTI compared the complete genome sequence of HAdV and the serotypes 2,3,and 7 Hexon gene sequences,the conserved region including serotypes HAdV 2,3,7 were designed.While maintaining the common forward primers that had been reported,the reverse primers were redesigned with oligo7.0 to achieve the production size of real time fluorescence quantitative PCR(qPCR).(3)With the results of above comparation,we found the serotypes 2,3,and 7 non conservative regions to design probes respectively.(4)The qPCR primers and probes concentration,annealing temperature and sensitivity specificity were analyzed.(5)By using the established triplex qPCR method to detect 138 HAdV positive clinical specimens and sequencing by nested PCR method to confirmed.Result: This study successfully established a triplex qPCR method to detect HAdV2,3,and 7.The analytical sensitivity of the method was 10 copies/reaction for each of HAdV serotypes 2,3 and 7,and no cross-reaction with other common respiratory viruses or other HAdV serotypes.The coefficients of variation(CV)of intra-assay and inter-assay were between 0.6% to 3.6%.Total 138 previously-confirmed HAdV positive samples tested,the detection agreement between qPCR and nested PCR was 96.38%(133/138).Conclusion: The triplex qPCR method established in this study can be rapid,convenient and accurate for the simultaneous detection of HAdV serotypes 2,3,7,and has potential clinical application value.Part two The relationship between non-enteric adenovirus with diarrhea in children——a case control studyObjective: Enteric adenovirus serotypes 40 and 41 have confirmed to associated with diarrhea,non-enteric adenovirus connected with diarrhea remains to be confirmed.Some studies indicated non-enteric adenovirus can detected in the specimens of diarrhea.We performed a case control study to explore the relationship between non-enteric adenovirus and diarrhea.Methods: We collected 273 fecal samples from children with diarrhea and 361 fecal specimens from the inpatient children in the surgical department with no diarrhea about half month from children's hospital of Hebei province China.All the 634 samples were detected and quantified by qPCR.Meanwhile,the HAdV positive specimens were amplified by nested PCR then sequencing.Diarrhea group of non-enteric adenovirus positive specimens were screened for common diarrhea related pathogens,excluding others common pathogens interference.The risk factors of diarrhea in HAdV serotypes were calculated by odds ratio(OR).Results: The positive rate of HAdV in diarrhea group was 28.94%(79 /273),including serotype 40,41,3,2,1,5 and 57,with 40,41 and 3 types most common.The positive rate of HAdV in the control group was 7.20%(26/361),including serotypes 40,41,3,2,1,5,57,6 and 31 serotypes.Among the HAdV positive diarrhea group,91.14% were less than 3 years old and 65.38% were in the control group.There was no significant difference in viral load of HAdV serotype 41 between the two groups(Z=-0.157,P=0.875).Non-enteric adenovirus serotype 3 is a risk factor for diarrhea in children(OR=9.205,P<0.001).Conclusion: Infection of non-enteric adenovirus serotype 3 may cause diarrhea. |
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