The Roche LINEAR ARRAY® HPV Test*: Improved performance

over previous research prototypes

Amanda Shepard, Sean Boyle, Dmitriy Kosarikov, Ray Apple, Janet Kornegay
Roche Molecular Systems Inc., 1145 Atlantic Avenue, Alameda, CA 94501, USA

Introduction Results
● The Roche prototype line blot assay (LBA) has been used as a research assay for genotyping human ● In general, signals were more intense on strips tested with the LINEAR ARRAY HPV Test (Figure 2)
papillomavirus (HPV)1–3 ● After running both protocols in tandem, 85/112 specimens gave complete concordance among high-
● Reproducibility and sensitivity were improved, compared with the MY09/11 primer system, by using risk (HR) genotypes
standardized PGMY primer pools instead of degenerate primers2,4 ● More genotypes were detected using the LINEAR ARRAY HPV Test, compared with the Roche LBA
● Further refinement and standardization have been incorporated into the Roche LINEAR ARRAY® HPV (Figure 3)
Test under development (Figure 1) ● 134 HR HPV infections were observed with the LINEAR ARRAY HPV Test, compared with 110 HR
● The improved protocol includes: HPV infections with the previous research protocol
– An integrated PCR master mix formulation, including uracil-N-glycosylase for the prevention of ● Repeat testing of the discrepant samples using an equivalent specimen input volume (5µL) increased
carry-over contamination the inter-assay concordance to 91% (102/112)
– Optimized TC9700 thermalcycling conditions ● The discordance could not be explained by the difference in specimen input volume for 37% of
– Increased specimen input discrepant samples (10/27)
● This new system has been designed to fulfill the clinical requirements for sensitivity, reliability and ● Results with probe 3X5X, using the LINEAR ARRAY HPV Test protocol, suggest type 52-positivity in
reproducibility, and provide validated and useful data on HPV status sample 0018 (Table 2 and Figure 2)

LINEAR ARRAY HPV Test Roche LBA

HPV genome β-globin
UNG digestion

Biotin-labeled Biotin-labeled PCR

PGMY09/11 β-globin
primer pools primer pair
High β-globin
Low β-globin

Denature and
hybridize
HPV 31

HPV 11
HPV 16

Add avidin-HRP Reference line

conjugate

Figure 2. A more intense signal and more genotypes were detected with the LINEAR ARRAY HPV Test
HPV-positive sample result Wash Denatured amplicon added to
Develop color membrane-bound oligonucleotide probe protocol

HPV: Human papillomavirus; UNG: Uracil-N-glycosylase; PCR: Polymerase chain reaction; HRP: Horseradish peroxidase Table 2. Positive probe results for 10 samples
Sample Initial testing Repeat testing
Figure 1. LINEAR ARRAY HPV Test ID LINEAR ARRAY Roche LBA LINEAR ARRAY Roche LBA
HPV Test probe hits probe hits HPV Test probe hits probe hits
(50 µL sample input) (5 µL sample input) (5 µL sample input) (5 µL sample input)

Objective 0013 53 53 – –
0015 68 68 – –
● To compare results from the LINEAR ARRAY HPV Test with those from the most commonly used 0016 39, 59 39, 59 – –
Roche LBA protocol 0017 6 6 – –
0018 16, 3X5X, 58(w), 59(w), 6 16, 3X5X, 6 3X5X, 6 3X5X, 6

Materials and methods 0019 3X5X, 56, 58, 6 3X5X, 56, 58, 6 – –
0024 3X5X, 58 3X5X, 58 – –
● DNA was extracted from 112 clinical specimens collected in PreservCyt® preservative solution, using a 0026 62, 83 83 – –
modified QIAGEN QIAamp MinElute™ procedure 0027 59, 66 66 66 66
● All specimens had previously been tested with the Digene Hybrid Capture® 2 (hc2) test, and yielded 0028 16, 3X5X, 58, 68 16, 3X5X, 58 16, 3X5X, 58, 68 16
positive results w: Weak signal
● The PCR DNA extract input volumes for the LINEAR ARRAY HPV Test and the Roche LBA protocols
were 50µL and 5µL, respectively
● Thermalcycling profiles are shown in Table 1 Conclusions
● Since the 3X5X probe cross-reacts with types 33, 35, 52 and 58, results were confirmed with additional
● These data suggest that the improved assay has increased sensitivity for genotyping
probes for types 33, 35 and 58
studies
Table 1. TC9700 and TC9600 thermalcycling profiles used in this study ● The ability to detect a broad spectrum of genotypes reliably and sensitively using the
LINEAR ARRAY HPV Test may allow:
TC9700 profilea for the LINEAR ARRAY HPV Test No. of cycles
– More accurate assignment of HPV infections as high-risk or low-risk
1 Hold 50ºC/2 min 1
– Better assessment of the role of persistent HPV infection in the natural history of
2 Hold 95ºC/9 min 1
3 Cycle 95ºC/30 s; 55ºC/1 min; 72ºC/1 min 40 cervical cancer
4 Hold 72ºC/5 min 1 – The significance of co-infection to be determined
5 Hold 72ºC/Indefinitely
6 Method LINK 1-2-3-4-5
TC9600 profile for the Roche LBA No. of cycles
1 Hold 95ºC/9 min 1 References
2 Cycle 95ºC/30 s; 55ºC/1 min; 72ºC/1 min 40
3 Hold 72ºC/5 min 1 1. Van Doorn LJ, Quint W, Kleter B et al. J Clin Microbiol 2002;40:979–83
4 Hold 4ºC/Indefinitely 2. Gravitt PE, Peyton CL, Alessi TQ et al. J Clin Microbiol 2000;38:357–61
5 Method LINK 1-2-3-4 3. Gravitt PE, Peyton CL, Apple RJ et al. J Clin Microbiol 1998;36:3020–7
aThe
TC9700 profile was run on MAX mode with all three cycling ramp speeds adjusted to 50% 4. Coutlee F, Gravitt P, Kornegay J et al. J Clin Microbiol 2002;40:902–7

20 LINEAR ARRAY HPV Test data

Number of positive samples

Roche LBA data

15

10

0
a b
16 18 26 31 33 35 39 45 51 52 53 56 58 59 66 67 68 69 70 73 82 9 6 11 40 42 54 55 61 62 64 71 72 81 83 84 08
IS3 61
cp
a
Variant of HPV-82; bHPV-89 HPV genotype

Figure 3. More genotypes were detected using the LINEAR ARRAY HPV Test protocol *Under development