基于毛细电泳的多重PCR方法检测儿童呼吸道腺病毒感染常见血清型的价值

The value of multiplex PCR method based on capillary electrophoresis in detecting common serotypes of respiratory adenovirus infection in children

目的建立一种呼吸道腺病毒常见血清型检测方法,并利用该方法检测温州地区儿童呼吸道人腺病毒(HAdV)感染的主要血清型。方法利用基于毛细电泳的多重PCR法建立呼吸道腺病毒常见12种血清型检测方法,将2018年1月至2019年12月收治于温州医科大学附属育英儿童医院,经直接免疫荧光法检测HAdV阳性的1059例急性呼吸道感染患儿的呼吸道标本作为研究对象,对其进行回顾性分析,采用多重PCR方法测定HAdV-1、2、3、4、5、7、14、21、37、40、41和55型等12种血清型,同时随机抽取部分标本与一代测序法比较检测结果一致性。结果共收集到1059份HAdV抗原阳性的呼吸道标本,多重PCR法检测发现,1种血清型阳性947例(89.4%),2种血清型混合感染13例(1.2%),阴性24例(2.3%),阳性但不能分型75例(7.1%)。947例单种血清型阳性儿童中,B亚群的HAdV-3型415例(43.8%),HAdV-7型318例(33.6%),HAdV-55型12例(1.2%),HAdV-21型2例(0.2%);C亚群的HAdV-2型108例(11.4%),HAdV-1型70例(7.4%),HAdV-5型16例(1.7%);E亚群的HAdV-4型6例(0.6%)。未检出HAdV-14、HAdV-37、HAdV-40和HAdV-41。随机抽取多重PCR法检测的HAdV分型阳性的标本51例,与一代测序的检测进行比较,均符合。结论建立并验证了基于毛细电泳的多重PCR法对儿童呼吸道腺病毒感染常见血清型的诊断价值。B亚群的HAdV-3、HAdV-7和C亚群的HAdV-2、HAdV-1为温州地区儿童呼吸道腺病毒感染的主要血清型,HAdV-14、HAdV-37、HAdV-40和HAdV-41未检出。

Objective To establish a common method for detecting serotypes of respiratory adenovirus,and to detect the main serotypes of respiratory human adenovirus(HAdV)infection in children in Wenzhou area.Methods A multiplex PCR method based on capillary electrophoresis was developed to detect 12 common serotypes of respiratory adenovirus.A total of 1059 children with acute respiratory infection who were admitted to Yuying Children′s Hospital Affiliated to Wenzhou Medical University from January 2018 to December 2019 with positive infection of HAdV detected by the direct immunofluorescence method were recruited and retrospectively analyzed.Multiplex PCR was performed to determine 12 serotypes of respiratory adenovirus,including HAdV-1,2,3,4,5,7,14,21,37,40,41 and 55.Meanwhile,some samples were randomly selected to examine the consistency in the detection result by the first-generation sequencing method.Results A total of 1059 specimens of respiratory secretions with positive HAdV antigen were collected.Detected by multiplex PCR method,947 cases(89.4%)were positive for 1 serotype,13 cases(1.2%)were mixed infection with 2 serotypes,and 24 cases(2.3%)were negative.In addition,75 cases(7.1%)were positive but could not be serotyped.Among the 947 children with the positive infection of a single serotype,415 cases(43.8%)were HAdV-3 in subgroup B,318 cases(33.6%)were HAdV-7,12 cases(1.2%)were HAdV-55,2 cases(0.2%)were HAdV-21,108 cases(11.4%)were HAdV-2 in subgroup C,70 cases(7.4%)were HAdV-1,16 cases(1.7%)were HAdV-5,and 6 cases(0.6%)were HAdV-4 in subgroup E.HAdV-14,HAdV-37,HAdV-40 and HAdV-41 were not detected.A total of 51 positive samples of HAdV infection detected by multiplex PCR were randomly selected to compare with the detection result by the first-generation sequencing,which were all consistent.Conclusions This study successfully established a multiplex PCR based on capillary electrophoresis in diagnosing common serotypes of respiratory adenovirus infection in children.HAdV-3,HAdV-7 of subgroup B and HAdV-2 and HAdV-1 of subgroup C were the main serotypes of respiratory adenovirus infection in children of Wenzhou area.HAdV-14,HAdV-37,HAdV-40 and HAdV-41 were not detected.