In addition, HPV-positivity was assessed by low-stringency Southern blot analysis of PCR products with a cocktail probe of HPV-specific DNA fragments.
In addition, EIA-negative samples were tested by low-stringency Southern blot analysis of PCR products with a cocktail probe of HPV-specific DNA fragments to assess whether HPV types were present that were not represented in EIA oligoprobe cocktails.
Southern blot hybridisation was performed under low-stringency conditions (Tm = -35 degrees C) using a whole genomic HPV 6/16/18 probe mixture and under high stringency conditions (Tm = -18 degrees C) with the whole genomic probes of HPV 16 and 33.
However, the probe for detection of HPV-52 amplicons in the LA-HPV test is a cross-reactive probe that also hybridises with types 33, 35, and 58.
To detect the 1 2 copies of HPV 16 in SiHa cells HPV probe at a concentration of 1 ng/μl was sufficient.
First, all DNA extracts were tested by using the Linear Array HPV Genotyping Assay (Linear Array; Roche Diagnostics, Indianapolis, IN, USA) and a HPV-52-specific PCR to resolve ambiguous positive results from the XR probe of the Linear Array HPV test (20 ).
Although sensitivity is the most important factor governing HPV detection on ICC due to the usual very low viral load in these cases, other technical caveats may contribute to false negative results, like HPV DNA variability on primers and probe binding sites and partial deletions of HPV genes integrated into cellular genome, among other factors.
In addition, HPV types that were not included in Probe B cocktail of HC II or unknown types of HPV can be detected using the PCR method.
Detection of HPV type 52 was based on a probe which is also known to signal in the presence of HPV types 33, 35 and 58.
Presence of HPV DNA, was determined by a probe hybridization technique that contains a mixture of HPV specific probes recognizing at least 54 mucosal HPV genotypes in a microtiter plate format (DNA Enzyme Immuno Assay -DEIA).
The hc2 test is based on the hybridization of HPV DNA with RNA specific probes and they hybrid detected by a chemiluminescent assay.
