Saturday, May 16, 2009

Hybrid Capture 2 Technology

Hybrid Capture 2 (hc2) technology serves as the platform for QIAGEN’s nucleic acid hybridization assay for detection of human papillomavirus (HPV), Chlamydia trachomatis (CT), and Neisseria gonorrhoea (GC). This diagnostic method allows rapid, standardized testing of genetic material of the infectious agents in virtually any laboratory setting. It employs specific RNA probes, hybridization, antibody capture, and signal amplification that utilizes qualitative chemiluminescent detection.


The digene HPV Test, using Hybrid Capture 2 technology, provides an accurate, cost-effective, user-friendly method by achieving reliable detection without the need for dedicated and isolated lab space, sophisticated laboratory expertise, or concern related to inhibition and contamination.
Benefits of Hybrid Capture:

* Same-day, objective results using a micro-plate test format
* High-throughput automation for handling increasing test volumes
* Minimal required specimen preparation allowing samples to be processed quickly and efficiently
* Less complex workflow than target amplification methods
* Reduced risk of cross-over contamination due to signal amplification instead of target amplification
* Extensive clinical validation data available

hc2 technology superior to PCR methods for HPV DNA testing

For routine HPV diagnostics, it is essential to have a test system at hand which reports clinically relevant results and, hence, can impact clinical decision-making. In contrast to the detection of other infectious disease parameters, a high clinical sensitivity is critical for reliable HPV diagnostics. The hc2 technology fulfills this requirement better than any other method as outlined in multiple clinical validation studies.

In contrast, a high analytical sensitvity as provided by nucleic acid amplification technologies such as PCR is counterproductive for HPV diagnostics as they may detect many transient HPV infections of clinical insignificance. Cervical cancer screening methods require HPV detection correlating to disease and not to the sheer presence of the virus, since 90% of infections is cleared by itself without consequences.

hc2 technology measures sensitivity versus defined clinical endpoints (CIN 3+/SCC) and ensures reporting of positive results only when risk of disease progression exists. QIAGEN’s digene HPV Test has an excellent clinical sensitivity of up to 100% in primary adjunctive screening when combined with a Pap smear test.

* Clinically validated cut-off of 5000 copies/ml
* Proven in extensive multiple trials
* Ensures high negative predictive value

PCR screening can miss severe disease


PCR methods show a high analytical sensitivity with detection limits between 10-400 copies of HPV DNA resulting in detection of clinically irrelevant viral levels as such infections can be cleared off by immune response. Due to viral integration and loss of the targeted regions (L1, E1, E2) or mutation, PCR can miss cases of severe precancer and cancer cases.

* Risk of missing cases of cancer
* Detection of clinically irrelevant viral levels


Hybrid capture (hc2) versus PCR — analytical sensitivity and clinical sensitivity. The analytical sensitivity of hc2 has been adjusted to 5,000 copies of HPV DNA, and the analytical sensitivity of PCR methods can be <10 copies of HPV DNA.
How Hybrid Capture 2 Technology works

The Hybrid Capture 2 System is a signal-amplification assay that uses a technique combining antibody capture and chemiluminescent signal detection. The basic steps of the Hybrid Capture 2 assay are outlined below:

Basic Steps

Clinical specimens are prepared by adding a base solution, which disrupts the virus or bacteria and releases target DNA. Presence or absence of target DNA is read from the results of the chemiluminescent reaction.

Hybridize RNA probe with target DNA Hybrid Capture Signal Amplification

Fig 1: Hybridize RNA probe with target DNA. Target DNA combines with specific RNA probes, creating RNA:DNA hybrids


Fig. 2: Hybrid Capture. RNA:DNA hybrids are captured onto a solid phase coated with universal capture antibodies specific for RNA:DNA hybrids.


Fig. 3: Signal Amplification. Capture RNA:DNA hybrids are detected with multiple antibodies conjugated to alkaline phosphatase. The signal resulting from the chemiluminescent reaction is read and results interpreted.

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