| abstract |
Discloses a novel detection and typing method for viruses, such as human papillomaviruses, based on real-time PCR using self-probing amplicon fluorescent primers. The method comprises: (IA) contacting the sample with a self-probing amplicon (‘virus self-probing amplicon’) comprising (i) a virus primer capable of hybridising to at least one target viral nucleic acid sequence and undergoing amplification thereof under primer amplification conditions to form a virus primer extension product; (ii) a virus probe comprising a nucleic acid sequence complementary to a target sequence of the virus primer extension product and capable of hybridisation thereto, provided that the self-probing amplicon is adapted to ensure that the virus probe is unresponsive to amplification under the primer amplification conditions; and (iii) a member of a virus signalling system, which system is capable of causing a detectable signal to be effected on hybridisation of the virus probe sequence to the virus primer extension product, whereby presence or absence of the target viral nucleic acid sequence in the sample is indicated by the detectable signal; (IB) amplifying the product of step (IA) under the primer amplification conditions to an extent enabling the detectable signal to be effected after step(II); and (II) separating the virus primer extension product from the target viral nucleic acid sequence; allowing the virus probe to hybridise to the target sequence of the virus primer extension product; and monitoring the signalling system. This method is quick, simple, specific, sensitive, and capable of estimating viral load per cell. The results of over 100 HPV typing reactions performed on cell lines, biopsies and cervical cytobrush samples are given which, when compared to the current reference HPV detection and typing technique, present a kappa value of 0.89. The method is also applicable to other viruses, such as SV40. |