Evaluation of a Modified Reverse Line Blot Assay for Detection
Evaluation of a Modified Reverse Line Blot Assay for Detection
Detection and typing of human papillomaviruses (HPVs) are requested by clinicians with growing frequency as part of the overall management of patients. A new method using consensus L1 amplification and a reverse line blot hybridization detection method has been described for broad spectrum HPV genotype identification. This method involves hybridization at 53°C in a shaking water bath, which many laboratories might find cumbersome. We describe a modified hybridization step, using a dry-air incubator, that simplifies the detection protocol. Overall, comparable results were obtained by the modified, more easily performed protocol, indicating that laboratories might validate and use this modified method.
Based on molecular and in vitro epidemiologic evidence, it is clearly evident that certain genotypes of human papillomavirus (HPV) are the principal causes of invasive cervical cancer. HPV-16 and HPV-18 are the 2 most prevalent types worldwide, accounting for more than 70% of cervical cancers. Owing to the presence of a large number of genotypes (>100), typing of HPV has proven to be expensive and time-consuming, often involving only a few selected HPV types. A broad-spectrum amplification system, in conjunction with a single hybridization and reverse line blot (LB) detection method, has been reported for simultaneous HPV genotype identification of up to 27 types. This assay involves hybridization of PCR amplicons to specific HPV probes immobilized on nylon strips and a subsequent LB stringent washing step in a water bath equilibrated to 53°C. We considered that these steps might be difficult and cumbersome for many laboratories and endeavored to adapt the water-bath system to a dry-air incubator system.
Detection and typing of human papillomaviruses (HPVs) are requested by clinicians with growing frequency as part of the overall management of patients. A new method using consensus L1 amplification and a reverse line blot hybridization detection method has been described for broad spectrum HPV genotype identification. This method involves hybridization at 53°C in a shaking water bath, which many laboratories might find cumbersome. We describe a modified hybridization step, using a dry-air incubator, that simplifies the detection protocol. Overall, comparable results were obtained by the modified, more easily performed protocol, indicating that laboratories might validate and use this modified method.
Based on molecular and in vitro epidemiologic evidence, it is clearly evident that certain genotypes of human papillomavirus (HPV) are the principal causes of invasive cervical cancer. HPV-16 and HPV-18 are the 2 most prevalent types worldwide, accounting for more than 70% of cervical cancers. Owing to the presence of a large number of genotypes (>100), typing of HPV has proven to be expensive and time-consuming, often involving only a few selected HPV types. A broad-spectrum amplification system, in conjunction with a single hybridization and reverse line blot (LB) detection method, has been reported for simultaneous HPV genotype identification of up to 27 types. This assay involves hybridization of PCR amplicons to specific HPV probes immobilized on nylon strips and a subsequent LB stringent washing step in a water bath equilibrated to 53°C. We considered that these steps might be difficult and cumbersome for many laboratories and endeavored to adapt the water-bath system to a dry-air incubator system.
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