PCR in situ hybridisation detection of HPV 16 in fixed CaSki and fixed SiHa cell lines.
OBJECTIVE
To investigate the feasibility of using fixed cells with the polymerase chain reaction (PCR) in situ hybridisation and to investigate possible reasons for reaction failure.


METHODS
Fixed SiHa and CaSki cells were used in an experimental model of PCR in situ hybridisation for the detection of low and intermediate copy number viral infection in fixed cells.


RESULTS
PCR in situ hybridisation was able to detect one to two copies of human papillomavirus (HPV) 16 in SiHa cells, using small fragment amplicons (120 base pairs), confirming the high detection sensitivity and flexibility of the technique. Problems were encountered with localisation of PCR amplified product in CaSki cells (200-300 copies of HPV 16 per cell) owing to diffusion of product post amplification. Overall, 40% of reactions were successful, which confirms the current unreliability of the technique. Within cell preparations, about 50% of cells contained amplified product.


CONCLUSIONS
PCR in situ hybridisation represents the marriage of two revolutionary molecular pathological techniques. However, it is currently unreliable, with reaction failure common. Standardised, dedicated equipment is urgently required if the technique is to achieve universal acceptance. In the future, the technique may be used to detect chromosomal translocations in human tumours and to study cellular gene expression.
Nuffield Department of Pathology and Bacteriology, University of Oxford.
J J
JJ
O'Leary
G
G
Browne
M I
MI
Johnson
R J
RJ
Landers
M
M
Crowley
I
I
Healy
J T
JT
Street
A M
AM
Pollock
F A
FA
Lewis
A
A
Andrew
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008969:Molecular Sequence Data; D027383:Papillomaviridae / Q000235:genetics*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016707:Tissue Fixation
D017931:DNA Primers; D004279:DNA, Viral