METHODS IN LABORATORY INVESTIGATION - IN-SITU AMPLIFICATION OF MEASLES-VIRUS RNA BY THE SELF-SUSTAINED SEQUENCE REPLICATION REACTION

BACKGROUND: The self-sustained sequence replication (3SR) reaction is an isothermal method for nucleic acid amplification that has several f eatures that make it an attractive alternative to PCR. We have studied the feasibility of the in situ 3SR reaction in cells using a measles virus-infected cell line as a model. EXPERIMENTAL DESIGN: The study wa s carried out in four steps. First, using RNA extracted from a measles -infected Vero Green monkey kidney cell Line, conditions for the in vi tro amplification of a segment of the nucleocapsid portion of the RNA viral genome were optimized for 420- and 119-bp 3SR products, and the results were compared. Second, 3SR was performed on intact infected ce lls in suspension, and the amount of RNA product was compared with inf ected cells without 3SR. Then, the 3SR reaction was conducted on cytos pin preparation slides, followed by in situ hybridization for detectio n of the amplification product. Finally, 3SR was carried out on sectio ns of formalin-fixed, paraffin-embedded cells, and the degree of ampli fication as detected by ISH was quantified and compared between infect ed cells with and without 3SR reaction. RESULTS: Specific amplificatio n of measles was observed in each of these types of preparations with an 8.5-fold rate of amplification in paraffin sections of formalin-fix ed cells (a mean of 272.5 +/- 65.3 grains/cell after 3SR amplification in comparison to 31.97 +/- 4.2 grains/cell without amplification). CO NCLUSIONS: A significant amount of amplification of RNA is possible wi th in situ 3SR (IS-3SR) and, in combination with ISH, offers several a dvantages compared with in situ PCR (IS-PCR), such as ease of use, lac k of conditions that lead to cell damage, and a specificity for RNA am plification. This is the first report of specific amplification of RNA within cells using the IS-SSR procedure, a technique that has a wide range of potential applications in pathology and molecular biology.