Kogawa K, Nakata S, Ukae S, Adachi N, Numata K, Matson D O, Estes M K, Chiba S
Department of Pediatrics, Sapporo Medical University School of Medicine, Japan.
Arch Virol. 1996;141(10):1949-59. doi: 10.1007/BF01718206.
A dot blot hybridization assay was developed for detection of human calicivirus/Sapporo/82/J (HuCV/Sa/82) or strains closely related to HuCV/Sa/82 in stool specimens. The cDNA derived from the RNA-dependent RNA polymerase (RDRP) region of HuCV/Sa/82 was used as a positive probe and the pBR322 DNA as a negative control probe. Both probes were labeled with digoxigenin and the products of hybridization reaction were detected with an anti-digoxigenin antibody-alkaline phosphatase conjugate. This assay was specific for HuCV/Sa/82 and for HuCV antigenically related to HuCV/Sa/82. The lower limit of sensitivity of this assay was estimated to be about 10(5) physical particles or 10 pg of cDNA, similar to that of the previously developed ELISA for HuCV. In 1273 stool specimens obtained from children with acute gastroenteritis in Sapporo, Japan, 110 (8.6%) contained small round structured viruses by EM and 23 (1.8%) were positive for HuCV antigenically related to HuCV/Sa/82 by either the hybridization assay or ELISA. A higher positive rate was obtained with the dot blot assay (21%) than by ELISA (10%), suggesting that the dot blot assay either detects HuCV more broadly than the ELISA or detects HuCV covered with fecal antibodies which interrupt antigen-antibody reactions in the ELISA. Negative results for detection of Norwalk virus (NV) cDNA and feline calicivirus (FCV) RNA by both this assay and the ELISA indicated that the HuCV/Sa/82 strain is distinct antigenically and genetically from NV and FCV.
开发了一种斑点印迹杂交试验，用于检测粪便标本中的人杯状病毒/札幌/82/J（HuCV/Sa/82）或与HuCV/Sa/82密切相关的毒株。来自HuCV/Sa/82的RNA依赖性RNA聚合酶（RDRP）区域的cDNA用作阳性探针，pBR322 DNA用作阴性对照探针。两种探针均用地高辛标记，杂交反应产物用抗地高辛抗体-碱性磷酸酶偶联物检测。该试验对HuCV/Sa/82以及与HuCV/Sa/82抗原相关的HuCV具有特异性。该试验的灵敏度下限估计约为10(5)个物理颗粒或10 pg cDNA，与先前开发的HuCV ELISA相似。在从日本札幌的急性胃肠炎儿童中获得的1273份粪便标本中，110份（8.6%）通过电子显微镜检查含有小圆结构病毒，23份（1.8%）通过杂交试验或ELISA检测出与HuCV/Sa/82抗原相关的HuCV呈阳性。斑点印迹试验（21%）获得的阳性率高于ELISA（10%），这表明斑点印迹试验要么比ELISA更广泛地检测到HuCV，要么检测到被粪便抗体覆盖的HuCV，而这些粪便抗体在ELISA中会干扰抗原-抗体反应。该试验和ELISA检测诺如病毒（NV）cDNA和猫杯状病毒（FCV）RNA均为阴性结果，表明HuCV/Sa/82毒株在抗原性和基因上与NV和FCV不同。
