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Reliability of the Roche AMPLICOR® Human Papillomavirus (HPV) Test

Kurt Beyser, Ulrike Schön, Andreas Lindauer
Life Science Department, Synlab, Medizinisches Fachlabor, Weiden, Germany

Introduction

Persistent infection with high-risk human papillomavirus (HPV) genotypes is a necessary cause of cervical carcinoma; HPV testing offers a way to improve cervical screening programs and decrease cancer mortality1–4 PCR-based HPV DNA assays, such as the Roche AMPLICOR® HPV Test (Figure 1), have the advantage of being extremely sensitive and robust, and can be performed on very small aliquots of cervical samples in liquid cytology media, and on samples from archival paraffin-embedded blocks The issue of possible cross-contamination with previously amplified products (amplicon) must be resolved for PCR-based HPV testing to be clinically reliable The AMPLICOR HPV Test prevents cross-contamination, by incorporating deoxyuridine, resulting in a product susceptible to degradation by AmpErase® enzyme (uracil-Nglycosylase), contained within the master mix We performed multiple assays with the AMPLICOR HPV Test, to assess the level of crosscontamination
Native HPV genome UNG digestion Denature and hybridize PCR

HPV-positive sample

HPV-negative control

PCR-positive control

Figure 3. AMPLICOR HPV Test amplification tray/microwell layout Table 1. AMPLICOR HPV Test PCR conditions using a PE9700 thermocyclera
Wash, add avidin-HRP conjugate

Biotinylated primers

Pre-incubation with UNG TaqGold activation (inactivation of UNG) Denaturation Annealing of biotinylated primers to target Extension by TaqGold polymerase Hold
aProgramming

50°C for 2 minutes 95°C for 9 minutes

HPV probe Immobilized hybrids react with avidin-HRP
TMB Oxidized Oxidized TMB TMB TMB

Hybridization

95°C for 30 seconds 54°C for 45 seconds 40 cycles 72°C for 30 seconds

HRP produces color upon addition of TMB substrate

Add stop solution, read absorbance

HPV: Human papillomavirus; UNG: Uracil-N-glycosylase; PCR: Polymerase chain reaction; HRP: Horseradish peroxidase; TMB: 3,3',5,5'-tetramethylbenzidene

Figure 1. AMPLICOR HPV Test

72°C, 1hb
ramp rate: 50%, ramp speed: max, volume: 100 µL; bSamples to be removed within 1 hour; UNG: Uracil-N-glycosylase

Objective

To assess the level of cross-contamination after multiple assays using the AMPLICOR HPV Test

Results Materials and methods
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Protocols were carried out in three separate areas of the laboratory (Figure 2) A total of five AMPLICOR HPV Test runs, each consisting of 12 replicates of HPV type 16positive samples (5 x 105 SiHa cells/mL) and 12 HPV-negative controls [HPV(-)C], were tested HPV-positive cultured cells were spiked into PreservCyt® preservative solution DNA extraction was performed using the QIAGEN QIAamp MinElute™ Media kit procedure The beta-globin (BG) gene is isolated concurrently, and assesses cellular adequacy, extraction and amplification for each individually processed specimen HPV(+) and HPV(-)C samples were alternated (Figure 3) on the MinElute™ vacuum manifold and on the amplification tray, in order to assess potential carry-over PCR was performed on the GeneAmp® PCR System 9700, as shown in Table 1 Amplicons were then hybridized in parallel in a microwell plate format against HPV probes (strips 1–4) and BG probes (strips 5–8) The cross-contamination rate was defined as the ratio of the number of false-positives to the total number of negative samples tested
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The HPV cross-contamination rate was zero; 0/60 HPV(-)C samples had HPV OD450 values ≥0.2 A low level of human DNA contamination was observed in one negative sample (BG OD450 of 0.308 [cut-off, 0.2]), which was declared a false-positive resulting from a technical error All 60 HPV(+) cell samples were positive for HPV and BG (HPV and BG OD450 ≥1.0) No HPV(+) or BG OD450 readings of ≤0.2 were observed, indicating that no significant sample loss occurred during preparation

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Conclusions

The AMPLICOR HPV Test is highly sensitive and reliable, with no crosscontamination of HPV DNA observed when tested with high levels of DNA input

One sample showed a low level of BG contamination; however, this may be due to technical error since hybridization of the same sample against HPV in the multiplex test was negative

Pre-PCR area

PCR area

Post-PCR area

A) DNA extraction B) PCR reagent setup (master mix) C) Sample added to amplification tubes PCR steps as shown in Table 1

Denaturation Washing Color development Plate reading

References
1. Walboomers JM, Jacobs MV, Manos MM et al. J Pathol 1999;189:12–9 2. Bohmer G, van den Brule AJ, Brummer O et al. Am J Obstet Gynecol 2003;189:118–20 3. Petry KU, Bohmer G, Iftner T et al. Am J Obstet Gynecol 2002;186:28–34

Figure 2. Partition of work into laboratory areas

4. Munoz N, Bosch FX, de Sanjose S et al. N Engl J Med 2003;348:518–27