Berra A, Dutt J E, Foster C S
Hilles Immunology Laboratory, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, USA.
Cornea. 1996 Jan;15(1):55-61.
The purpose of our study was to develop a method for detecting herpes simplex virus (HSV) DNA that combined the high sensitivity of the polymerase chain reaction (PCR) with the precise anatomical localization provided by in situ hybridization (ISH). We used in situ PCR (ISPCR), ISH, and standard PCR methods to determine the proportion of Vero cells carrying HSV-1-specific DNA before and after 1, 2, and 4 h of infection with HSV-1 or with HSV-2. Uninfected Vero cells and Vero cells infected with HSV-2 were never found to be positive for HSV-1 DNA by either ISPCR, ISH, or PCR. In contrast, using ISPCR, HSV-1 infected Vero cells showed an increase in the percentage of cells containing HSV-1 DNA from 20% at 1 h to 76% at 4 h after infection. Comparing the ISPCR results with ISH and standard PCR demonstrated that ISPCR was markedly more sensitive than ISH; in fact, the sensitivity of in situ PCR was similar to that seen with standard PCR. These results demonstrate that ISPCR is a highly sensitive method for amplifying genomic DNA sequences within intact single cells. This technique combines the exquisite sensitivity of conventional PCR technology with the precise cellular localization afforded by ISH. In addition, it allows for an accurate quantitative determination of the number of virally infected cells.
我们研究的目的是开发一种检测单纯疱疹病毒（HSV）DNA的方法，该方法将聚合酶链反应（PCR）的高灵敏度与原位杂交（ISH）提供的精确解剖定位相结合。我们使用原位PCR（ISPCR）、ISH和标准PCR方法，来确定用HSV-1或HSV-2感染1、2和4小时前后，携带HSV-1特异性DNA的Vero细胞比例。通过ISPCR、ISH或PCR，未发现未感染的Vero细胞以及感染HSV-2的Vero细胞对HSV-1 DNA呈阳性。相比之下，使用ISPCR，感染HSV-1的Vero细胞显示，感染后1小时含HSV-1 DNA的细胞百分比从20%增加到4小时后的76%。将ISPCR结果与ISH和标准PCR进行比较表明，ISPCR比ISH明显更灵敏；事实上，原位PCR的灵敏度与标准PCR相似。这些结果表明，ISPCR是一种用于扩增完整单细胞内基因组DNA序列的高灵敏方法。该技术将传统PCR技术的极高灵敏度与ISH提供的精确细胞定位相结合。此外，它还能对病毒感染细胞的数量进行准确的定量测定。
