荧光定量PCR用于日本血吸虫感染高危环境早期预警的研究

Study on early warning of high risk environment of Schistosoma japonicum infection by quantitative real-time PCR

采用实时荧光定量PCR(qRT-PCR)法检测感染早期哨鼠内脏组织日本血吸虫DNA,探索最佳检测时间,以达到早期监测预警血吸虫感染高危环境的目的。取20只阳性钉螺置于25℃温水中1 h逸出血吸虫尾蚴。将昆明小鼠随机分为自然感染组(6只)、定量感染组(36只)和阴性对照组(9只)。自然感染组小鼠转移至含有血吸虫尾蚴水体的笼内模拟野外现场自然感染1 h,定量感染组小鼠经腹部贴片法感染尾蚴(40±5)条/鼠,阴性对照组小鼠不作感染。自然感染组小鼠感染后第1、2、5天随机解剖2只,定量感染组小鼠感染后第1、2、3、5、7、14天随机解剖6只,阴性对照组小鼠同步随机剖检1只。取各组小鼠腹部皮肤和心、肺、肝组织,提取DNA后进行qRT-PCR检测血吸虫DNA,记录循环阈值(Ct值),判断各组小鼠血吸虫感染情况;定量感染组小鼠还需单独检测个体各时间段肺组织中血吸虫DNA的Ct值。qRT-PCR检测结果显示,自然感染组小鼠腹部皮肤组织中血吸虫DNA在感染后第1、2天检测结果为阳性,肺组织在感染后第1、2、5天检测结果均为阳性,肝组织和心脏组织在感染后第5天为阳性。定量感染组小鼠皮肤在感染后第1、2、3天检测结果为阳性,肺组织在感染后所有时间点均为阳性,肝组织在感染后第2~14天均为阳性,心脏组织在感染后第3天和第7天为阳性,血液样品在感染后第5天为阳性。定量感染组小鼠个体肺组织在感染后第2~5天检测结果为阳性。提示哨鼠感染早期对其肺组织进行qRT-PCR检测血吸虫DNA,具有早期预警血吸虫感染高危环境的价值。

To rapidly monitor and warn high-risk environments with Schistosoma japonicum,detecting S.japonicum DNA in the visceral tissues of sentinel rats at the early stage of infection by quantitative real-time PCR(qRT-PCR).20 infected Oncomelania hupensis were placed in 25℃warm water to release cercariae.The rats(KM species)were divided into three groups randomly,i.e.Group A with natural cercarial infection,Group B with quantitative cercarial infection and Group C without cercarial infection as a negative control group.In Group A,6 rats were transferred into the cage with the water with cercariae for 1 h,which imitate the natural infection field.36 rats in Group B were infected with cercariae(40±5 per mouse)via the abdominal skin route.9 rats in Group C were not infected with cercariae.The rats in Group A were anatomized randomly on day 1,2 and 5 after infection.The rats in Group B were anatomized randomly on day 1,2,3,5,7 and 14 after infection,respectively.The rats in Group C were randomly selected to anatomized synchronously with the Group A and B.The tissues of abdominal skin,heart,lung and liver of each rat were collected after the dissection.Then the genomic DNA of the total collected tissues was extracted and tested for schistosome DNA through qRT-PCR.The Ct values were recorded to determine the infection rates in each group,especially for Group B,in which each rat's DNA Ct value of the lung tissue needed to record at different time period.As the qRT-PCR results showed,schistosome DNA in Group A was detected in lung on day 1,2 and 5 after infection,and in liver on day 5 after infection.While in group B,the schistosome DNA was detected in the lung tissue on day 1,2,3,5,7 and 14 after infection,and later in the liver tissue on day 2,3,5,7 and 14.It showed that the schistosome DNA in lung in group B can be detected from the 2nd to the 5th day after infection.The results suggested that the detection of schistosome DNA in the lung tissue by qRT-PCR at the early stage of sentinel rat infection has the value for early monitoring and warning of the high-risk environment with schistosome infection.