Figure 51-19 Smear of cervical cells stained with probe for papillomavirus DNA. Dark-staining cells contain virus DNA. (Courtesy Children's Hospital Medical Center of Akron, Akron, Ohio.)
and detect RNA viruses by using the enzyme reverse transcriptase (RT). The first step in RT-PCR includes making a complementary DNA strand of the RNA segment in question. The usual PCR steps used to multiply the DNA target are then performed, leading to DNA amplicons whose identification signifies the presence of the original RNA sequence. The rapid appearance and broad application of molecular diagnostics will require the introduction and use of standardized materials and external quality control programs. In addition, the use of universal internal controls throughout the procedure will further ensure accuracy.
Conventional Cell Culture. Viruses are stria intracellular parasites, requiring a living cell for multiplication and spread. To detect virus using living cells, suitable host cells, cell culture media, and techniques in cell culture maintenance are necessary. Host cells, re-
Figure 51 -20 Real-time PCR detection of heipes simplex virus (HSV). Black, red, and light green lines represent three different HSV-1 viruses. Pink and dark green lines represent two different HSV-2 viruses. A, Cycle crossover detection of HSV-1 and HSV-2 amplicons, with all viruses detected between cycles 34 and 40. B, Melt curve confirmation of the presence of HSV-1 and HSV-2 viruses. HSV-1 amplicons melt at approximately 54° C (three HSV-1 viruses confirmed) and HSV-2 amplicons melt at approximately 68" C (one HSV-2 virus confirmed).
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