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 features 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.


METHODS
The study was carried out in four steps. First, using RNA extracted from a measles-infected Vero Green monkey kidney cell line, conditions for the in vitro 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 cells in suspension, and the amount of RNA product was compared with infected cells without 3SR. Then, the 3SR reaction was conducted on cytospin preparation slides, followed by in situ hybridization for detection of the amplification product. Finally, 3SR was carried out on sections of formalin-fixed, paraffin-fixed, paraffin-embedded cells, and the degree of amplification as detected by ISH was quantified and compared between infected cells with and without 3SR reaction.


RESULTS
Specific amplification of measles was observed in each of these types of preparations with an 8.5-fold rate of amplification in paraffin sections of formalin-fixed cells (a mean of 272.5 +/- 65.3 grains/cell after 3SR amplification in comparison to 31.97 +/- 4.2 grains/cell without amplification).


CONCLUSIONS
A significant amount of amplification of RNA is possible with in situ 3SR (IS-3SR) and, in combination with ISH, offers several advantages compared with in situ PCR (IS-PCR), such as ease of use, lack of conditions that lead to cell damage, and a specificity for RNA amplification. This is the first report of specific amplification of RNA within cells using the IS-3SR procedure, a technique that has a wide range of potential applications in pathology and molecular biology.
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002522:Chlorocebus aethiops; D005784:Gene Amplification; D005814:Genes, Viral; D017403:In Situ Hybridization / Q000379:methods*; D008459:Measles virus / Q000235:genetics*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000032:analysis / Q000235:genetics; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics*; D014709:Vero Cells
D009841:Oligonucleotides; D012367:RNA, Viral