Objective To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses(HEVs)from clinical samples and to contribute to etiological surveillance of HEV-related diseases.Methods A panel...Objective To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses(HEVs)from clinical samples and to contribute to etiological surveillance of HEV-related diseases.Methods A panel of RT-nPCR assays,consisting of published combined primer pairs for VP1 genes of HEV A–C and in-house designed primers for HEV-D,was established in this study.The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID50 perμL and copies perμL,and the newly established methods were tested in clinical specimens collected in recent years.Results The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID50 perμL and 10 virus copies perμL,and for the complete VP1 gene was 1 CCID50 perμL and 100 virus copies perμL,using serially-diluted virus stocks of five serotypes.As a proof-of-concept,25 serotypes were identified and complete VP1 sequences of 23 serotypes were obtained by this system among 858 clinical specimens positive for HEVs during the past eight surveillance seasons.Conclusion This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A–D,providing rapid,sensitive,and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.展开更多
【目的】探明引起安徽某鸭场雏鸭肝脏出血和大量死亡的病原及其遗传进化特征。【方法】对安徽省某鸭场的病死雏鸭中采集的出血肝脏开展鸭已知病原核酸检测、病原分离鉴定和动物回归试验,在明确其病原为鸭3型甲肝病毒(Duck hepatitis A v...【目的】探明引起安徽某鸭场雏鸭肝脏出血和大量死亡的病原及其遗传进化特征。【方法】对安徽省某鸭场的病死雏鸭中采集的出血肝脏开展鸭已知病原核酸检测、病原分离鉴定和动物回归试验,在明确其病原为鸭3型甲肝病毒(Duck hepatitis A virus type 3,DHAV-3)的基础上分析其VP1基因序列分子特征。【结果】细菌分离结果显示,未分离到细菌;经病毒核酸(RT-)PCR检测结果显示,鸭3型甲肝病毒(DHAV-3)核酸阳性,未检测出其他已知引起鸭肝出血的病毒核酸。将该阳性样品经鸭胚进行病毒分离与传代,发现接种后鸭胚发生死亡,胚体全身出血,对第5代尿囊液经RT-PCR检测为DHAV-3,将其命名为AH230225。经测定,该分离株的鸭胚半数致死量(Effective lethal dose 50,ELD_(50))为10^(−4.17)/0.1 mL。动物回归试验表明,该毒株对樱桃谷雏鸭的致死率为80%,且攻毒死亡鸭肝脏和肾脏的剖检病变与临床典型病变相近。对该分离毒的VP1基因核苷酸序列进行同源性分析,显示AH230225株的VP1基因核苷酸序列与AH07株DHAV-3(安徽分离株)的同源性最高,为98.8%,与GenBank登录的10株DHAV-3分离株VP1基因核苷酸序列同源性为90.4%~98.8%,而与DHAV-1和DHAV-2的VP1基因核苷酸序列同源性分别为62.1%~63.0%、64.6%~64.9%;基于VP1蛋白氨基酸序列的遗传进化显示,该分离株与AH07株DHAV-3处于同一小进化分支上,亲缘关系最近;而与SD01株、G株和韩国株(AP-04009、AP-03337)等亲缘关系较远,即远离DHAV-1和DHAV-2进化分支。【结论】引起安徽某鸭场雏鸭肝脏出血和大量死亡的病原为鸭3型甲肝病毒DHAV-3,同时明确了该毒株VP1基因的分子特征及遗传进化规律,为深入研究DHAV-3的致病机制和制定防控措施提供科学依据。展开更多
The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to...The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to detecting antibody of foot-and-mouth disease virus serotype A. Western-Blot test showed that the VP1 recombinant protein could be used as detective antigen as it can be specifically recognized by bovine positive serum of FMDV serotype A. By employing matrix titra- tion method, the optimal parameters were obtained as follows： 1 mg/L VP1 protein as coating antigen, Vserum：Vblocking solution = 1：50 dilution for serum and Vsecondary enzyme-linked antibedies：Vblocking solution ---1：2 000 for enzyme combined antibodies. The results showod that the sensitivity and specificity of this method were 94.32% and 99.09% respectively, the coefficients of variations in intra-assay and inter-assay reproducibility tests was lower than 8%. Compared with liquid phase blocking ELISA kits, the agreement of 201 serum samples reached 92.54%. The VP1-ELISA method established here is specific, sensitive, stable and simple, which can be used to monitor the antibody level of FMD serotype A.展开更多
基金National Science and Technology Major Projects[No.2017ZX10104001 and No.2017ZX10103008]Fujian Provincial Natural Science Foundation[No.2016J01350]。
文摘Objective To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses(HEVs)from clinical samples and to contribute to etiological surveillance of HEV-related diseases.Methods A panel of RT-nPCR assays,consisting of published combined primer pairs for VP1 genes of HEV A–C and in-house designed primers for HEV-D,was established in this study.The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID50 perμL and copies perμL,and the newly established methods were tested in clinical specimens collected in recent years.Results The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID50 perμL and 10 virus copies perμL,and for the complete VP1 gene was 1 CCID50 perμL and 100 virus copies perμL,using serially-diluted virus stocks of five serotypes.As a proof-of-concept,25 serotypes were identified and complete VP1 sequences of 23 serotypes were obtained by this system among 858 clinical specimens positive for HEVs during the past eight surveillance seasons.Conclusion This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A–D,providing rapid,sensitive,and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.
文摘【目的】探明引起安徽某鸭场雏鸭肝脏出血和大量死亡的病原及其遗传进化特征。【方法】对安徽省某鸭场的病死雏鸭中采集的出血肝脏开展鸭已知病原核酸检测、病原分离鉴定和动物回归试验,在明确其病原为鸭3型甲肝病毒(Duck hepatitis A virus type 3,DHAV-3)的基础上分析其VP1基因序列分子特征。【结果】细菌分离结果显示,未分离到细菌;经病毒核酸(RT-)PCR检测结果显示,鸭3型甲肝病毒(DHAV-3)核酸阳性,未检测出其他已知引起鸭肝出血的病毒核酸。将该阳性样品经鸭胚进行病毒分离与传代,发现接种后鸭胚发生死亡,胚体全身出血,对第5代尿囊液经RT-PCR检测为DHAV-3,将其命名为AH230225。经测定,该分离株的鸭胚半数致死量(Effective lethal dose 50,ELD_(50))为10^(−4.17)/0.1 mL。动物回归试验表明,该毒株对樱桃谷雏鸭的致死率为80%,且攻毒死亡鸭肝脏和肾脏的剖检病变与临床典型病变相近。对该分离毒的VP1基因核苷酸序列进行同源性分析,显示AH230225株的VP1基因核苷酸序列与AH07株DHAV-3(安徽分离株)的同源性最高,为98.8%,与GenBank登录的10株DHAV-3分离株VP1基因核苷酸序列同源性为90.4%~98.8%,而与DHAV-1和DHAV-2的VP1基因核苷酸序列同源性分别为62.1%~63.0%、64.6%~64.9%;基于VP1蛋白氨基酸序列的遗传进化显示,该分离株与AH07株DHAV-3处于同一小进化分支上,亲缘关系最近;而与SD01株、G株和韩国株(AP-04009、AP-03337)等亲缘关系较远,即远离DHAV-1和DHAV-2进化分支。【结论】引起安徽某鸭场雏鸭肝脏出血和大量死亡的病原为鸭3型甲肝病毒DHAV-3,同时明确了该毒株VP1基因的分子特征及遗传进化规律,为深入研究DHAV-3的致病机制和制定防控措施提供科学依据。
基金Supported by Joint Funds of the NSFC and Henan Province(U1204327)Henan Provincial Key Laboratory Construction(122300413217)
文摘The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to detecting antibody of foot-and-mouth disease virus serotype A. Western-Blot test showed that the VP1 recombinant protein could be used as detective antigen as it can be specifically recognized by bovine positive serum of FMDV serotype A. By employing matrix titra- tion method, the optimal parameters were obtained as follows： 1 mg/L VP1 protein as coating antigen, Vserum：Vblocking solution = 1：50 dilution for serum and Vsecondary enzyme-linked antibedies：Vblocking solution ---1：2 000 for enzyme combined antibodies. The results showod that the sensitivity and specificity of this method were 94.32% and 99.09% respectively, the coefficients of variations in intra-assay and inter-assay reproducibility tests was lower than 8%. Compared with liquid phase blocking ELISA kits, the agreement of 201 serum samples reached 92.54%. The VP1-ELISA method established here is specific, sensitive, stable and simple, which can be used to monitor the antibody level of FMD serotype A.
