Hukkanen V, Rehn T, Kajander R, Sjöroos M, Waris M
Department of Virology, University of Turku, FIN-20520 Turku, Finland.
J Clin Microbiol. 2000 Sep;38(9):3214-8. doi: 10.1128/JCM.38.9.3214-3218.2000.
We have introduced a time-resolved fluorometry (TRF)-based microwell hybridization assay for PCR products in detection of herpes simplex virus (HSV) in cerebrospinal fluid (CSF) specimens. TRF is a sensitive nonradioactive detection technique which involves the use of lanthanide chelates as fluorescent labels. We used PCR primers from the glycoprotein D genes of HSV type 1 (HSV-1) and HSV-2. The biotinylated PCR products were collected on streptavidin-coated microtitration wells and hybridized with short oligonucleotide probes, europium labeled for HSV-1 and samarium labeled for HSV-2. The TRF results were obtained as counts per second and as signal-to-noise (S/N) ratios. The sensitivity of the assay was 0.1 infectious units (PFU) of HSV in CSF specimens, and the S/N values increased with the virus amount, up to 68.5 for 10(3) PFU of HSV-1 and to 58.5 for 10(3) PFU of HSV-2, allowing semiquantitation of HSV in CSF. The primers and probes recognized all the studied 48 HSV wild-type samples, with S/N ratios of 12.4 to 190 (HSV-1) and 5.1 to 248 (HSV-2). We tested CSF specimens, 100 for each HSV type, which were HSV PCR negative by Southern blot and 22 CSF specimens which were HSV-1 or -2 PCR blot positive. In the TRF test, the mean S/N ratio for the HSV-1-negative CSF was 1.37 (standard deviation [SD] = 0.513) and for the HSV-2-negative CSF it was 1.03 (SD = 0.098). The HSV-1 blot-positive CSF yielded S/N ratios of 3.6 to 85.9, and the HSV-2 blot-positive CSF yielded ratios from 1.9 to 13. Using the mean S/N ratio for negative CSF specimens + 3 SD as the cutoff yielded all the previously HSV-positive specimens as TRF positive. The TRF PCR assay for HSV in CSF specimens is a rapid and sensitive method, improves interpretation of PCR results, and is well suited for automation.
我们引入了一种基于时间分辨荧光法（TRF）的微孔杂交检测法，用于检测脑脊液（CSF）标本中的单纯疱疹病毒（HSV）的PCR产物。TRF是一种灵敏的非放射性检测技术，它使用镧系螯合物作为荧光标记。我们使用了来自1型单纯疱疹病毒（HSV-1）和2型单纯疱疹病毒（HSV-2）糖蛋白D基因的PCR引物。生物素化的PCR产物收集在链霉亲和素包被的微量滴定孔上，并与短寡核苷酸探针杂交，用铕标记HSV-1，用钐标记HSV-2。TRF结果以每秒计数和信噪比（S/N）表示。该检测法的灵敏度为脑脊液标本中0.1个感染单位（PFU）的HSV，S/N值随病毒量增加，HSV-1的10(3) PFU时高达68.5，HSV-2的10(3) PFU时高达58.5，从而可对脑脊液中的HSV进行半定量。引物和探针识别了所有研究的48个HSV野生型样本，S/N比为12.4至190（HSV-1）和5.1至248（HSV-2）。我们检测了每种HSV类型各100份脑脊液标本，这些标本通过Southern印迹法检测HSV PCR为阴性，以及22份脑脊液标本，这些标本HSV-1或-2 PCR印迹为阳性。在TRF检测中，HSV-1阴性脑脊液的平均S/N比为1.37（标准差[SD]=0.513），HSV-2阴性脑脊液的平均S/N比为1.03（SD=0.098）。HSV-1印迹阳性的脑脊液S/N比为3.6至85.9，HSV-2印迹阳性的脑脊液S/N比为1.9至13。以阴性脑脊液标本的平均S/N比+3 SD作为临界值，可使所有先前HSV阳性的标本在TRF检测中呈阳性。脑脊液标本中HSV的TRF PCR检测法是一种快速灵敏的方法，可改善PCR结果的解读，且非常适合自动化。
