Van Dyke R B, Murphy-Corb M
Department of Pediatrics, Tulane University, New Orleans, Louisiana 70112.
J Clin Microbiol. 1989 Aug;27(8):1739-43. doi: 10.1128/jcm.27.8.1739-1743.1989.
We have developed an RNA-cDNA hybridization assay for the detection of respiratory syncytial virus (RSV) RNA in nasopharyngeal samples. We chose to use as probe a cDNA complementary to the nucleocapsid protein gene of RSV, integrated into the plasmid vector pBR322. The lower limit of sensitivity of the assay is 8.2 X 10(2) PFU of the Long strain of RSV. In throat washes with added cell-free virus, the assay can detect 3.3 X 10(3) PFU of RSV. Respiratory secretions were collected from a group of 104 infants in New Orleans, and 73 of the samples were tested for RSV by immunofluorescence (IF). All were then frozen at -70 degrees C for later testing by hybridization, and 67 were tested for RSV antigens by enzyme immunoassay (EIA). A second set of respiratory secretions from 48 infants in Denver were cultured for virus, assayed for RSV antigen by EIA, and then frozen for later testing by hybridization. For those samples on which IF was performed, hybridization, compared with IF, had a sensitivity of 49% and a specificity of 66%. For samples tested by EIA, hybridization had a sensitivity of 60% and a specificity of 81% compared with EIA. Compared with virus isolation, hybridization assay had a sensitivity of 73% and a specificity of 92%. With clinical samples, the sensitivity and specificity of the assay were improved with the addition of a control blot, which was hybridized to the plasmid vector (pBR322). The performance of the hybridization assay can be expected to improve when the assay is used with fresh clinical material rather than frozen samples.
我们开发了一种RNA - cDNA杂交检测法，用于检测鼻咽样本中的呼吸道合胞病毒（RSV）RNA。我们选择使用与RSV核衣壳蛋白基因互补的cDNA作为探针，该cDNA整合到质粒载体pBR322中。该检测法的灵敏度下限为RSV长株的8.2×10² PFU。在添加无细胞病毒的咽洗液中，该检测法可检测到3.3×10³ PFU的RSV。从新奥尔良的一组104名婴儿中收集呼吸道分泌物，其中73份样本通过免疫荧光（IF）检测RSV。所有样本随后在-70℃冷冻，以备稍后通过杂交检测，67份样本通过酶免疫测定（EIA）检测RSV抗原。从丹佛的48名婴儿中收集的第二组呼吸道分泌物进行病毒培养，通过EIA检测RSV抗原，然后冷冻以备稍后通过杂交检测。对于进行了IF检测的那些样本，与IF相比，杂交的灵敏度为49%，特异性为66%。对于通过EIA检测的样本，与EIA相比，杂交的灵敏度为60%，特异性为81%。与病毒分离相比，杂交检测法的灵敏度为73%，特异性为92%。对于临床样本，通过添加与质粒载体（pBR322）杂交的对照印迹，该检测法的灵敏度和特异性得到了提高。当该检测法用于新鲜临床材料而非冷冻样本时，预计其性能会有所改善。
