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J Clin Microbiol. Jul 2006; 44(7): 2428–2433.
PMCID: PMC1489532

Assessment of MagNA Pure LC Extraction System for Detection of Human Papillomavirus (HPV) DNA in PreservCyt Samples by the Roche AMPLICOR and LINEAR ARRAY HPV Tests

Abstract

Roche Molecular Systems recently released two PCR-based assays, AMPLICOR and LINEAR ARRAY (LA), for the detection and genotyping, respectively, of human papillomaviruses (HPVs). The manual specimen processing method recommended for use with both assays, AmpliLute, can be time-consuming and labor-intensive and is open to potential specimen cross-contamination. We evaluated the Roche MagNA Pure LC (MP) as an alternative for specimen processing prior to use with either assay. DNA was extracted from cervical brushings, collected in PreservCyt media, by AmpliLute and MP using DNA-I and Total Nucleic Acid (TNA) kits, from 150 patients with histologically confirmed cervical abnormalities. DNA was amplified and detected by AMPLICOR and the LA HPV test. Concordances of 96.5% (139 of 144) (κ = 0.93) and 95.1% (135 of 142) (κ = 0.90) were generated by AMPLICOR when we compared DNA extracts from AmpliLute to MP DNA-I and TNA, respectively. The HPV genotype profiles were identical in 78.7 and 74.7% of samples between AmpliLute and DNA-I or TNA, respectively. To improve LA concordance, all 150 specimens were extracted by MP DNA-I protocol after the centrifugation of 1-ml PreservCyt samples. This modified approach improved HPV genotype concordance levels between AmpliLute and MP DNA-I to 88.0% (P = 0.043) without affecting AMPLICOR sensitivity. Laboratories that have an automated MP extraction system would find this procedure more feasible and easier to handle than the recommended manual extraction method and could substitute such extractions for AMPLICOR and LA HPV tests once internally validated.

Molecular and in vitro epidemiological evidence have established a definitive association of various human papillomavirus (HPV) genotypes with the development of cervical cancer and its precursor lesions, cervical intraepithelial neoplasia 2/3 (CIN2/3) (2, 3, 5, 20, 21, 30). Cervical cancer is the second most common cancer of women worldwide, with approximately half a million new cases diagnosed and more than 270,000 deaths occurring annually (3, 10, 19, 20, 22, 31).

There are more than 100 different HPV genotypes identified within the Papillomavirus family, of which approximately 40 are known to infect the anogenital mucosa (4, 9). Based on their implicated etiological role in the carcinogenesis of the cervix, genital HPV genotypes are subdivided into associated risk types, namely, low-risk (LR) and high-risk (HR) types. Although both groups of virus type can result in abnormal cell growth, only the HR types may lead to the development of cervical cancer (3, 20, 21, 30). Nearly all cases of cervical cancer are caused by infection, with at least one of the 13 accepted HR “oncogenic” HPV types (i.e., types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) (2, 5, 21, 30). The majority of HPV infections are transient and asymptomatic and are often cleared by the host immune system without leading to cervical abnormality, yet persistent infection with a HR-type substantially increases the risk of CIN2/3 and potential development of carcinoma of the cervix (7, 13, 26, 31).

The application of molecular techniques for the detection of HPV in clinical specimens have been evaluated and clinical algorithms for patient care are continually being devised. In most evaluations, detection of HPV DNA has been by the signal amplification assay Hybrid Capture 2 (HC2; Digene Corp., Gaithersburg, MD) (6, 25, 29) and target amplification methods such as PCR, although many of these have largely constituted in-house assays. Commonly used PCR-based HPV detection methods have included the use of the general primer pairs MY09/11 and their derivatives PGMY09/11 (11, 12), GP5+/GP6+ (8, 14), and the SPF10 system (16, 23, 24). Only recently have broad-spectrum PCR-based assays become commercially available, although they are not yet approved by the U.S. Food and Drug Administration for clinical use. HC2 is the only commercially available, U.S. Food and Drug Administration-approved HPV test for primary screening, in conjunction with a Pap smear, of women 30 years of age or older, and triage of women of any age with atypical cells of undetermined significance (“ASCUS”) pap results (Digene). Roche Molecular Systems recently released two assays for the amplification and detection of HPV DNA. The first, the AMPLICOR HPV test, is a qualitative in vitro test for the detection of the 13 HR genotypes described earlier. These types are the same as those detected by the HC2 assay. The second is the LINEAR ARRAY (LA) HPV genotyping test, a qualitative test for the detection and typing of up to 37 HPV mucosal genotypes (including the 13 HR types present in the AMPLICOR HPV test). Current specimen processing protocols for both assays recommend the use of manual extraction of DNA using the AmpliLute liquid media extraction kit, based on the QIAamp method (QIAGEN, Inc., Valencia, CA). This method of DNA preparation is time-consuming and labor-intensive and is prone to potential specimen cross-contamination, particularly when large numbers of specimens are being processed. An alternative method for DNA extraction, already in use with several other molecular detection assays, is the automated MagNA Pure LC (MP) extraction system. We evaluated here the utility of the automated MP for the extraction of DNA from 150 cervical brush specimens (in PreservCyt media) in place of the current manual AmpliLute method for HPV testing by both AMPLICOR and LA assays.

MATERIALS AND METHODS

Clinical specimens.

Cervical brush specimens were collected from 150 women undergoing ablation treatment for histologically confirmed cervical abnormality at the Royal Women's Hospital, Melbourne, Australia. All cervical brushings were rinsed into ThinPrep vials containing PreservCyt transport medium prior to routine laboratory testing by the Digene HC2 assay for HR HPV genotype detection. The 150 specimens, collected between May 2001 and December 2002, were selected from a larger panel of specimens under the following criteria: HC2 negative (n = 75) or HC2 positive (n = 75), with relative light unit ratios of 1 to 5 (n = 20), >5 to 50 (n = 20), >50 to 500 (n = 20), or >500 (n = 15). Specimens within this range of relative light unit ratios were selected in order to compare the sensitivity of the AMPLICOR and LA assays using nucleic acid extracted from samples with low to high HPV viral loads.

Specimen preparation.

Initially, DNA was isolated from the PreservCyt samples using three different procedures: (i) a 250-μl aliquot was extracted by AmpliLute (QIAamp MinElute media kit) in conjunction with a QIAvac 24 Plus vacuum system, according to the manufacturer's instructions; (ii) a 200-μl aliquot was extracted by MP using the DNA-I kit (blood cells high-performance protocol); and (iii) a 200-μl aliquot was extracted by MP using the Total Nucleic Acid (TNA) kit. Nucleic acid was eluted into a final volume of 120 μl for AmpliLute extractions or 110 μl for MP extractions. In addition to these three extraction protocols, all 150 samples were re-extracted by using a modified procedure involving the centrifugation of 1-ml aliquots of the PreservCyt samples at 13,000 × g for 20 min prior to discarding of the supernatant. The resultant cell pellets were resuspended into 200 μl of sterile phosphate-buffered saline, which was extracted by MP, using the DNA-I kit, into a final volume of 100 μl.

After nucleic acid isolation, all samples were analyzed by both HPV AMPLICOR and LA assay (Roche Molecular Systems, Inc., Alameda, CA) for HPV detection and genotyping, respectively.

AMPLICOR HPV test.

The AMPLICOR HPV test involves PCR amplification of target DNA, followed by nucleic acid hybridization using a microwell plate system for the detection of 13 HR anogenital HPV genotypes (i.e., types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). This test utilizes the amplification of an approximate 165-bp region within the L1 gene of the HPV genome. In addition, this test incorporates the simultaneous amplification of a region within the human β-globin gene as a control for the cell adequacy, nucleic acid extraction, and amplification efficiency of individually processed specimens (11, 12). PCR was performed in a final reaction volume of 100 μl composed of 50 μl of AMPLICOR HPV master mix (Roche Molecular Systems) and 50 μl of isolated DNA, according to the manufacturer's recommendations. Importantly, when using DNA extracted from 1 ml of pelleted PreservCyt, only 10 μl of DNA was used (maintaining an equivalent amount of cells to the standard protocol), with 40 μl of sterile water added to achieve a reaction volume of 100 μl. Amplification was performed in a GeneAmp PCR System 9700 with gold block (Applied Biosystems, Foster City, CA) using the following parameters: 2 min at 50°C and 9 min at 95°C; followed by 40 cycles of: 95°C for 30 s, 54°C for 45 s, and 72°C for 30 s; followed by a final hold at 72°C. PCR amplicons were immediately denatured by the addition of 100 μl of AMPLICOR denaturation solution (Roche Molecular Systems) and incubation at room temperature for 10 min. A dual 96-microwell plate system for the simultaneous detection of β-globin and HPV was used for amplicon-to-probe hybridization. A total of 100 μl of the denatured PCR products was hybridized and detected using the standard AMPLICOR protocol. Absorbance readings of greater than 0.2 were classified as positive for either HPV and/or β-globin presence.

LA HPV genotyping test.

The LA HPV genotyping test involves PCR amplification of target DNA, followed by nucleic acid hybridization using a reverse line blot system for the simultaneous detection of up to 37 anogenital HPV genotypes (types 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39, and CP6108). The LA HPV genotyping test amplifies a region, of approximately 450 bp in length, within the L1 gene of the HPV genome. As with the AMPLICOR test, this assay simultaneously amplifies a region within the human β-globin gene as a control for cell adequacy, nucleic acid extraction, and PCR efficiency. PCR was performed in a reaction volume of 100 μl, using 50 μl of LA HPV master mix (Roche Molecular Systems) and 50 μl of DNA, as described previously. The cycle parameters used were as follows: 2 min at 50°C and 9 min at 95°C; followed by 40 cycles of 95°C for 30 s, 55°C for 1 min, and 72°C for 1 min; followed by a final extension at 72°C for 5 min and holding at 72°C for up to 4 h prior to denaturation. The PCR amplicons were denatured by the addition of 100 μl of LA denaturation reagent (Roche Molecular Systems), followed by incubation at room temperature for 10 min. The denatured amplicons (75 μl) were then hybridized and detected using the recommended LA protocol. The LA HPV genotyping strips were manually interpreted using the HPV reference guide provided.

Statistical analysis.

Statistical analyses for P values were performed using 2-×-2 contingency tables (http://www.graphpad.com/quickcalcs/contingency1.cfm), with two-tailed P values calculated by using the Fisher exact test. All P values of <0.05 were considered statistically significant. Concordance levels were measured by Cohen's kappa test (http://www.niwascience.co.nz/services/statistical/kappa). Concordance values of 0.21 to 0.40 were considered “fair,” values of 0.41 to 0.60 were considered “moderate,” values of 0.61 to 0.80 were considered “substantial,” and values of 0.81 to 1.00 were considered “almost perfect.”

RESULTS

Standard MagNA Pure LC extraction protocol.

The levels of sample adequacy, nucleic acid extraction, and amplification efficiency, among the 150 specimens, based on β-globin positivity and/or HPV positivity, differed marginally between extraction methods (Table (Table1).1). Sample adequacy was higher with MP extracts using either the DNA-I (99.3%) or the TNA kit (98.0%) than with the AmpliLute method (96.7%).

TABLE 1.
Adequacy, based on β-globin and/or HPV positivity, of 150 samples extracted by four methods and tested by the AMPLICOR HPV test

HPV positivity among the 145 valid AmpliLute-extracted samples were compared to equivalent extracts from MP DNA-I (n = 149) or TNA (n = 147). Overall, concordances of 96.5% (139 of 144; κ = 0.93) and 95.1% (135 of 142; κ = 0.90) were obtained by AMPLICOR HPV test when we compared the AmpliLute DNA extracts to those of DNA-I and TNA, respectively (Table (Table2).2). Six of the seven AmpliLute-negative, MP-positive samples were different specimens, of which four were confirmed as having an HR HPV genotype by LA. Of the five AmpliLute-positive, MP-negative samples, four were different specimens, of which three were confirmed by LA as having an HR HPV genotype. All of these confirmations were performed using LA genotyping results from AmpliLute extracted DNA, since this was both the standard method and had a tendency to generate a higher proportion of HPV genotypes than the MP DNA extracts: 211 genotypes in total (AmpliLute) compared to 178 genotypes (DNA-I) and 167 genotypes (TNA).

TABLE 2.
Comparison of the AMPLICOR HPV test using DNA extracted by AmpliLute with MagNA Pure LC DNA-I, TNA kit, or DNA-I (1 ml PreservCyt) extracts

Nucleic acid extracted by AmpliLute and MP DNA-I and TNA kits was evaluated by the HPV LA genotyping test. All 150 samples analyzed generated positive β-globin results using the three extraction methods, with signal intensities of the β-globin and HPV-specific bands being very similar between methods. Overall, HPV type profiles were identical in 118 (78.7%) and 112 (74.7%) of samples between AmpliLute and DNA-I and between AmpliLute and TNA, respectively (Table (Table3).3). Statistically, there was no difference in HPV-type profiles detected in the AmpliLute DNA extracts with those detected among the DNA-I or TNA extracts (P = 0.495). Of the 150 specimens, HPV type profiles ranged from single-type infections to multiple-type infections composed of up to seven HPV genotypes (data not shown). Single-HPV-type infections were identified in approximately one-third of the specimens (34.7 to 37.3%), with similar proportions identified as either multiple HPV infections or HPV LA-negative. Samples that exhibited discrepant HPV profiles between extraction protocols predominantly comprised an additional 1 to 2 types, 31 of 32 (96.9%) for AmpliLute/DNA-I and 37 of 38 (97.4%) for AmpliLute/TNA, of which the majority were extra types detected among AmpliLute DNA extracts (Table (Table3).3). Apart from these discrepancies, the HPV genotypes identified by LA were identical between the different DNA preparations.

TABLE 3.
Analysis of the LINEAR ARRAY HPV test using DNA extracted by AmpliLute with MagNA Pure LC DNA-I, TNA kit, or DNA-I (1 ml PreservCyt) extracts

Modified extraction protocol: use of 1 ml of PreservCyt.

To assess whether the LA genotyping concordance between MP- and AmpliLute-extracted DNA could be improved, all 150 specimens were re-extracted by MP using the DNA-I kit in conjunction with 1 ml of pelleted PreservCyt. This method improved the correlation between the AmpliLute and DNA-I LA results from 78.7 to 88.0% (Table (Table3).3). Of the 18 discrepant HPV type profiles, 16 were shown to have one additional genotype by the DNA-I (1 ml) extraction protocol, with 1 having an extra type by AmpliLute and 1 with a substituted HPV genotype profile (Table (Table3).3). Aside from these discrepancies, the HPV genotypes identified by LA were the same among the different DNA preparations. Unlike the lack of difference between HPV-type profiles detected in the AmpliLute DNA extracts with those among the DNA-I or TNA extracts (P = 0.495), there was a significant difference between HPV profiles detected among DNA extracts from 1 ml of pelleted PreservCyt and AmpliLute with those by DNA-I and TNA protocols (P = 0.043 and P = 0.005, respectively) (Table (Table33).

To ascertain whether the 1 ml of PreservCyt modified extraction protocol affected the AMPLICOR results, despite using an equivalent ratio of cells as per standard protocol, we tested the DNA extracts from 1 ml of PreservCyt by AMPLICOR HPV Test and compared the results with those previously tested. Importantly, five samples were removed from this analysis since these were both β-globin and HPV negative. The level of HPV positivity among the AmpliLute DNA extracts was compared with levels obtained with MP TNA, DNA-I, or 1-ml DNA-I extracts. A comparable concordance level of 95.9% (139 of 145; κ = 0.92) was obtained by using the AMPLICOR HPV test with DNA extracted from AmpliLute and the modified 1-ml DNA-I protocol compared to 96.5% (κ = 0.93) and 95.1% (κ = 0.90), respectively, for the DNA-I and TNA methods (Table (Table2).2). With these outcomes, we have proposed a new strategy for the processing of PreservCyt samples prior to HPV testing by Roche AMPLICOR and/or LA (Fig. (Fig.11).

FIG. 1.
Flow chart illustrating the recommended procedure for HPV testing of PreservCyt samples using the Roche AMPLICOR and LINEAR ARRAY HPV tests.

DISCUSSION

Newly developed PCR-based assays such as the AMPLICOR HPV test and LINEAR ARRAY genotyping test provide a standardized, consistent, and rapid means of HPV detection. Since the release of the AMPLICOR HPV test, there have been increasing reports on its application in clinical practice (17, 18, 28). It has been demonstrated that CIN2/3, and subsequent carcinoma of the cervix, is correlated with HR HPV-type specific persistence (1, 7, 13, 15, 26, 31), although most of the clinical correlation data has been generated by HC2 testing (25, 27, 29). Importantly, the Roche LA assay provides the capability of being able to identify up to 37 HPV genotypes within an individual specimen. With this capacity, it can be determined whether a recurrent HPV positive result is in fact due to the persistence of a specific HPV genotype, meaning a substantially increased risk of disease progression (13, 26, 30, 31).

The AMPLICOR and LA tests are both associated with a recommended manual DNA preparation protocol, the AmpliLute method, which can be time-consuming and labor-intensive. The use of an automated system for DNA preparation prior to HPV testing with either AMPLICOR and/or LA would greatly facilitate the Roche HPV assays, in terms of simplicity, time and labor efficiency, and sample accuracy. A recent report comparing the AMPLICOR HPV test to an INNO-LiPA HPV genotyping assay utilized an MP automated extraction system, using the TNA kit, for the isolation of nucleic acid for AMPLICOR HPV testing (28), although no comparison was made of the extraction efficiencies with the current Roche-recommended AmpliLute extraction procedure. The present study sought to evaluate and compare automated MP DNA extraction with the AmpliLute protocol, with the intention of substituting these extraction methods for use with both Roche HPV assays.

Among the 150 PreservCyt specimens analyzed here, 5 extracts from the recommended AmpliLute protocol were found to be invalid due to both a β-globin- and an HPV-negative result. In contrast, only one and three were found to be invalid after DNA-I and TNA extraction, respectively, indicating that DNA isolation via MP was as efficient, if not better, than the current manual AmpliLute extraction method. The marginal differences in sample adequacy could be due to variations in sampling. Comparison of the AMPLICOR HPV results obtained with AmpliLute DNA to those from either the DNA-I or the TNA MP extraction demonstrated high levels of agreement (96.5 and 95.1%, respectively), with κ values of >0.90. For the AMPLICOR HPV test, use of the Roche MP in conjunction with either DNA-I or TNA kit demonstrated similar outcomes to AmpliLute extraction. Therefore, the use of the Roche MP with DNA-I or TNA kits offers an automated approach, thereby providing a more rapid, efficient, and less-labor-intensive means for sample preparation and also significantly reducing the potential for sample cross-contamination.

Comparison of the LA genotype results obtained with AmpliLute DNA to those from the DNA-I or TNA MP extraction demonstrated moderate levels of similarity: 74.7% for TNA and 78.7% for DNA-I. There was no significant difference between DNA-I and TNA in terms of their similarity to AmpliLute LA genotyping results (P = 0.495). The discordant samples displayed mainly extra HPV genotypes among the AmpliLute DNA extracts. Comparing AmpliLute and DNA-I, 87.5% (28 of 32) of the discrepancies constituted extra genotypes by the former, with 27 of these representing 1 to 2 extra genotypes (comprising 45.9% HR types and 54.1% LR types). Of the remaining extracts, two comprised extra genotypes by MP DNA-I (both HR types), and the other two were substituted types. Comparison of AmpliLute and TNA shows that 94.7% (36 of 38) of the discrepancies also constituted extra genotypes by AmpliLute, with 35 of these representing 1 to 2 extra genotypes (comprising 36.6% HR types and 63.4% LR types) and 1 having an extra 4 genotypes (1 HR and 3 LR types). Of the remaining, one sample comprised an extra genotype by MP TNA (HR type), with the other being a substituted type. These results indicate that the current AmpliLute protocol for DNA preparation can detect more HPV genotypes than the MP DNA-I or TNA kits for HPV genotyping using the Roche LA HPV genotyping test.

However, in efforts to improve the level of LA correlation between AmpliLute- and MP-extracted DNA, all specimens were re-extracted by MP DNA-I using 1 ml of PreservCyt (rather than 200 μl as previously tested). It is important to note that for the AMPLICOR test, only 10 μl of the 100-μl eluate (rather than 50 μl) was used in the PCR, thereby maintaining an equivalent overall sample quantity to the recommended protocol. However, to maximize HPV detection by LA, 50 μl of eluate was used, which is equivalent to five times more sample. Using DNA obtained from this modified 1-ml approach, the correlation with AmpliLute LA results significantly improved from 78.7% for MP DNA-I to 88.0% for the MP DNA-I 1-ml protocol (P = 0.043). Importantly, DNA extracts from 1 ml of PreservCyt did not significantly affect the correlation with AmpliLute AMPLICOR results: 96.5% agreement between AmpliLute and MP DNA-I compared to 95.9% with the DNA-I 1-ml method. The modified 1-ml approach not only identified the extra genotypes identified by AmpliLute, previously undetected in the DNA-I extracts, but also identified a number of additional genotypes undetected in AmpliLute-extracted DNA. These additional genotypes identified through the processing of 1 ml of PreservCyt composed of nine HR and seven LR HPV types among 16 different samples. The nine HR types may likely prove important to clinical outcome, while the seven LR types may carry less clinical significance. However, identification of these additional genotypes may be valuable in the event of identifying HR HPV persistence, a key factor in cervical cancer development.

In the present study, we demonstrated that there is no significant difference in the outcome of the AMPLICOR HPV test when DNA extracted by the Roche MP is used as opposed to the recommended AmpliLute method, and thus the Roche MP method could substitute for these extractions. However, if testing by both AMPLICOR and LA is required, extraction by MP using a DNA-I kit of a 1-ml aliquot of PreservCyt pelleted and resuspended into 200 μl of PBS is recommended. The recommended manual DNA extraction protocol using the AmpliLute extraction kit, although providing adequate DNA quality, is more time-consuming and labor-intensive than other systems, such as the automated MP. Laboratories that possess an automated MP would find the extraction procedure much more simplified than the recommended manual AmpliLute method and could substitute such extractions for the AMPLICOR and LA HPV tests once internally validated. Although the present study evaluated the utility of the MagNA Pure LC, other automated nucleic acid purification platforms may provide similar improvements for the processing of specimens prior to HPV testing. However, assessment of such platforms would require further studies for comparison to the AmpliLute protocol prior to their use in HPV testing.

Acknowledgments

We thank Roche Molecular Systems for the provision of reagents.

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