WO2009116884A2

WO2009116884A2 - Human papillomavirus detection kit

Info

Publication number: WO2009116884A2
Application number: PCT/PT2009/000015
Authority: WO
Inventors: Rui Jorge GONÇALVES PEREIRA NOBRE; Teresa Maria Caldeira Martins; Luis Fernando Morgado Pereira De Almeida
Original assignee: Universidade De Coimbra; Centro De Neurociências E Biologia Celular; Instituto Português De Oncologia De Coimbra De Francisco Gentil, Epe
Priority date: 2008-03-18
Filing date: 2009-03-18
Publication date: 2009-09-24
Also published as: PT2009116884W; WO2009116884A3

Abstract

The present invention provides a method and a diagnostic kit for detection and typing of all human papillomavirus (HPV) comprising: nucleic acid amplification of a region of L1 gene using universal HPV primers, treating of the amplified fragment in four independent restriction reactions with different enzymes, namely Pstl. Haelll, Ddel and Rsal, and identification of HPV type by comparison of RFLP patterns with the reference patterns of known genotypes using an HPV genotyping scheme.

Description

Description Human Papillomavirus Detection Kit

Field of the invention

[1] The present invention relates to a methodology and a diagnostic kit for the detection and identification of all mucosal human papillomavirus (HPV). State-of-the-art

[2] Ii is presently well established and globally accepted that the persisting infection with a human papillomavirus (HPV) of high-risk type is the main cause of cervical cancer. HPVs are a heterogeneous group of double stranded, non-enveloped DNA viruses of the Papillomaviridae family. Al present, more than 100 HPV types have been identified worldwide, approximately 50 of which infecting the anogcnital epithelium. According to the recent classification from the International Agency for Research on Cancer (IARC), the mucosal HPV types are divided into four groups based on their oncogenic activity: high-risk (HPVs 16, 18, 31. 33. 35, 39, 45, 51, 52. 56, 58, and 59), probable high-risk (IlP Vs 26, 53. 66, 68, 73. 82), low-risk (HPVs 6. 11. 13, 40, 42. 43. 44. 54, 61, 70, 72, 81 and 89) and undetermined-risk types (HPVs 30, 32, 34, 62, 67, 69, 71. 74. 83. 84, 85. 86, 87, 90, 91). Due to the existing oncogenic differences between mucosatropic HPVs. it is clinically important not only to detect, but also to accurately identify different HPV types.

[3] H PV testing was firstly introduced to compensate the poor sensitivity and specificity of the Pap .smear cytology (Papanicolaou test). At the present time, in the majority of the cervical screening programs around the world, HPV-DNA is usually detected by a non-PCR-hased assay which consists on a signal-amplifying hybridization method, the Hyhrid Capture System Il (HClI) (Qiagcu Gailhersburg Inc., MD, USA, previously Digcne Corp.). This methodology (US4.849.332) allows the detection of 13 probable/ high-risk types and 5 low-risk types using two different cocktails of RNA probes, in two separate reactions.

[4] Although, being the only test approved by the Food and Drug Administration (FDA),

HC U presents important limitations: it does not provide information about the specific type of HPV that is detected, it does not detect all known probable/high-risk types, it is less sensitive than Polymerase Chain Reaction (PCR) methodologies and the cross- a activity of the two cocktail probes reduces the clinical relevance of a positive result.

[5] In order to improve the quality of HPV -detection, new methodologies have been developed, mainly carried out using the polymerase chain reaction (PCR). Genotyping of HPV can be done by type-specific PCR using primers that recognize only one specific type. An alternative approach is the use of universal primers, which are directed to a highly conserved region of rhe HPV LI gene and are virtually capable of detecting all mucosal HPV types in a single PCR reaction. The papillomaviruses are subsequently typed through analysis of the sequence of the amplified fragment by different methods, such as DNA sequencing, nucleic acid hybridization, and restriction fragment length polymorphism analysis (RFLP).

[6] PCR using consensus primers with subsequent restriction fragment length polymorphism analysis (PCR-RFLP) has been demonstrated to be a simple, sensitive and financially advantageous methodology to detect and characterize HPV-DNA in clinical specimens. However. PCR-RFLP assays previously described (Lungu et al. MoI Cell Probes. 1992, 6:145-152 - US5.814.448; Kay et al., J. Virol. Methods. 2002, 159:159-170; Naqvi et al. J. Virol. Methods. 2004, 117:91-95: Santiago et al. J. Clin. Virol. 2006, 25:89-97; WO2006/015991) present important limitations, since they often make use of a large number of restriction endonucleases or the set of selected enzymes do not allow the individual identification of all mucosal types and/or discrimination between high- and low-risk types. Furthermore, different HPV types usually present identical or similar RFLP patterns. In the documentWO2006/015991, although a reasonable discrimination between high- and low-risk types is achieved using a single restriction enzyme (CviRI), some HPV types still present the same RFLP patterns (for instance, HPV 11 and 30, and HPV 44 and 55). Moreover, several HPV types (HPV 18 and 68, HPV 18 and 70, HPV 18 and 42, HPV 31 and 58, HPV 33 and 13, HPV 51 and 83, HPV 56 and 11, HPV 58 and 62, HPV 82 and 53, HPV 6 and 64, HPV 61 and 84) are indistinguishable on agarose or polyacrylamide gels.

[7] The same is observed in the US5.814.448, where 3 restriction endonucleases (Haelll,

Rsal, and Pstl) are used together in a single-tube restriction reaction. In this assay, different types of HPV (for instance, HPV 18 and 45, HPV 30 and 53, HPV 66 and 74, HPV 82 and 83, HPV 82 and 85, HPV 73 and 106, and HPVs 31, 54 and 97) display identical or similar cleavage patterns.

[8] An additional disadvantage of all above-mentioned PCR-RFLP assays regards the complexity and time-consuming procedures associated with the interpretation of RFLP patterns and corresponding HPV type identification.

[9] The present invention aims:

- to provide a simple and cost-effective methodology for individual identification of all known and unknown mucosal HPV types, which can be performed using standard laboratorial equipment and in settings of poor financial resources;

- to provide the minimum number of restriction endonucleases that allow the complete and individual genotyping of all known mucosal HPV types;

- to provide an accurate and time-effective scheme for the interpretation of RFLP patterns and HPV type identification;

- to provide a diagnostic kit comprising all reagents for the amplification of the HPV DNA from a biological sample and subsequent RFLP analysis, an HPV genotyping scheme for an accurate interpretation of the results and instructions for using the kit. Brief description of the drawings

LlO] Figure 1 shows the sequences identification numbers of the primers used in the present invention.

[11] Figure 2 shows a scheme for mucosal HPV genotyping after RFLP analysis of MY09/11 amplicons using Pstl, HaelH, Ddel, and Rsal restriction endonucleases.

[ 12] Figure 3 shows a validation of RFLP restriction reactions on 10% polyacrylamide gel

(RFLP patterns from co-digestion of MY09/1 IPCR products of HPV 16 and Betal/2 amplicon). Asterisks indicate fragments from Betal/2 amplicon restriction. Lane Ml = 50 bp DNA molecular weight marker; Lane M2 = 25 bp DNA molecular weight marker.

[13] Figure 4 shows theRFLP patterns of MY09/11 PCR products of HPV 16 (a), HPV 58

(b) and HPV102 (c). Lane Ml = 50 bp DNA molecular weight marker; Lane P = undigested MY09/11 PCR amplicon; Lane M2 = 25 bp DNA molecular weight marker. description of the invention

[14] The present invention relates to a method and a diagnostic kit for detection and/or typing of all mucosal human papillomavirus (HPV).

[ 151 The method of the present invention comprises the following steps:

(I) Amplification of HPV DNA: DNA extracted from clinical samples is amplified using a set of universal primers, which are directed to highly conserved regions of the HPV Ll gene and are potentially capable of detecting all HPV types.

[16] Although the standard PCR was the preferred amplification method used in the present invention, mainly because it is a well-known, practical and cost-effective methodology, any of the other amplification methodologies known in the art can be performed (ligase chain reaction, transcription-based amplification system, or PCR using low-temperature ready-to-use moderately thermostable DNA polymerase system).

[17] In an embodiment of the present invention, universal primers MY09/MY 11 (SEQ ID

NO: 1 and 2) (Manos et al. Cold Spring Harbor Press. 1989, 7:209-214) or PGMY09/11 primer system (SEQ ID NO: 3 to 20) (Gravitt et al. J Clin Microbiol. 2000, 38:357-361) have been used.

[18] (H) Restriction analysis of amplified DNA : The resulting product of HPV DNA amplified in I) is subsequently analyzed in four independent restriction reactions, namely Ddel (5'-C/TNAG-3'), Pstl (5'-CTGCA/G-3'X HaeIII (5'-GG/CC-3') and Rsal (5'-GT/AC-3'). However, any other restriction endonucleases that recognize the same sequences (isoschizomers) can be used.

[19] (III) Identification of HPV type : The restrictions products obtained in II) are subsequently analyzed in agarose or polyacrylaraide gels pre-stained with a nucleic acid stain for DNA (EtBr. SYBR green, Gel star, etc) or using any other assay which have been proposed for DNA fragment size measurements.

[20] HPV type identification is performed by comparison of RFLP patterns obtained for each restriction enzyme with the reference RFLP patterns (Table 1).

[21] In the present invention, a genotyping scheme (Figure 2) was included, which allows the identification of HPV types in two easy steps: analyzing of occurrence/ non-occurrence of the Pstl, HaeIII Ddel and Rsal restriction reactions and subsequent comparison of the restriction fragments obtained by Rsal. Therefore, using this scheme, the RFLP patterns are subsequently analyzed in the following order: Pstl → HaeIII → Ddel -→ Rsal. At the end of the scheme, comparison is made between the restriction fragments obtained by Rsal restriction with the expected sizes defined for each HPV type.

[22] The present invention has resulted from an extensive in silico analysis of all known mucosal HPV DNA sequences for the presence of restriction endonuclease cleavage sites within the region of Ll that is amplified using universal MY primers, as follows: 1) A total of 296 DNA sequences deposited in GenBank, corresponding to 49 genital HPV types (6, 11, 13, 16, 18, 26, 30, 31, 32, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 97, 102, and 106) and 2 HPV subtypes (HPV 55 and 64) were aligned using the 'Alignment tool' of CLC Free Workbench program, version 3.2 (CLC bio, Denmark). Subsequently, DNA segments of the Ll gene that are bracketed by the MY09/11 primers were selected and compiled in a file A.

[23] 2) A file B was created, which included the set of following restriction en- donucleases: Pstl (5'-CTGCA/G-3'), HaeIII (5'-GG/CC-3'), Rsal (5'-GT/AC-3') and Ddel (5'-C/TNAG-3').

[24] This set of enzymes was pre-selected through an in silico analysis and allows identification of the 49 MY09/11 segments corresponding to the 49 reference HPV types.

[25] 3) Using the 'Restriction site analysis tool' of CLC Free Workbench program, the information of files A and B was crossed in order to predict the RFLP fragment sizes of each MY-amplicon restriction (Table 1).

[26] Table I:

[27] In table 1 , restriction fragments larger than 40 bp are in bold. Fragments under this size are not considered for discrimination of HPV types, because they are not visualized with good resolution in polyacrylamide or agarose gels. The superscript letters in Table 1 (a, b, and c) show HPV variants that have an altered RFLP-pattern with regards to their respective reference HPV type.

[28] The computer simulation showed that the use of Ddel, Haelll, Pstl, and Rsal, in four independent reactions, allows the complete genotyping of all mucosal HPV types. Each HPV type has a specific set of RFLP patterns, which is not shared by any other HPV type and can be clearly identified by visual discrimination in 10% polyacrylamide or in 3% agarose gels, except for one particular situation (case of HPV 85 and b variant of HPV 82), in which the size of the restriction fragments differed only by 9 bp. In this specific case, the problem can be solved using a 20% polyacrylamide gel, using type-specific PCR or by sequencing this MY09/11 amplicon.

[29] 4) Finally, based on the 74 sets of RFLP-patterns obtained for each HPV type (Table 1) and in the fact that each restriction enzyme does not cut some of MY09/11 amplicons, a genotyping scheme was developed.

[30] In Figure 2, where this genotyping scheme is defined, each box represents a restriction reaction using a specific enzyme. Draw lines represent the possible results when restriction does not occur, whereas dashed lines represent the possible results when restriction occurs. In two particular situations (designated by 1 and 2), draw lines also represent the possible results when restriction reaction originates an RFLP- fragment larger than 420 bp.

[31] The HPV type is identified in two easy steps: (1) sequential analysis of occurrence/ non-occurrence of Pstl, Haelll, Ddel and Rsal restriction reactions and (2) subsequent comparison of the restriction fragments obtained by Rsal digestion with the expected sizes (in brackets) defined for each HPV type by in silico analysis.

[32] In cases in which Rsal digestion does not occur (groups B, C and H), or when two or more HPVs present similar Rsal-RFLP patterns (groups A, D, E, F, G, I), HPV type can be identified by comparison of the restriction fragment sizes obtained by Ddel or HaeIII restriction digestion ('confirmation pattern'). When a HPV type is identified, it is possible to confirm it, comparing all the RFLP-patterns obtained with data described in Table 1.

[33] One of the weak points of known HPV diagnostic assays is the appearance of false negatives, due to the poor quality of DNA or by the presence of PCR inhibitors. In the present invention, a control consisting of parallel amplification of the patient's own DNA is used to ensure the quality of DNA sample. Primers Beta 1 and Beta 2 (SEQ ID NO 21 and 22) were designed for amplification of a 501 bp fragment from beta-globin gene. However, any sequence fragment from human DNA can be used as target for this purpose.

[34] An additional control is performed to test the efficacy of each enzyme and to avoid the misidentification of types due to partial digestion, based in a parallel co-digestion of MY09/11 amplification products with the control DNA (product of beta-globin fragment amplification). Betal/2 amplicon is cleaved by the whole spectrum of selected restriction enzymes when optimal conditions are achieved, originating the following restriction fragments: Pstl: 322 bp + 179 bp; HaeIII: 279 bp + 222 bp; Ddel: 271 bp + 198 bp + 32 bp; Rsal: 251 bp + 210 bp + 40 (see Figure 3). The size of these fragments does not coincide with any of the fragments listed in Table 1.

[35] In conclusion, the present invention offers significant advantages, not only over the

PCR-RFLP techniques previously described, but also over other currently available HPV typing assays (like hybridization with type-specific probes, type-specific PCR, microarray methodologies): 1) it detects a higher number of HPV types (49 HPV types and 2 HPV subtypes); 2) it allows the individual detection of all known and unknown HPV types; and 3) it can be performed using standard laboratorial equipment and in settings of poor financial resources.

[36] Another aspect of the present invention also relates to an in vitro diagnostic kit for identification of HPV types. This kit comprises the following components:

- a standard nucleic acid amplification mix with universal HPV primers;

- 4 different restriction reaction vessels (each one contains a specific restriction enzyme and respective buffers) ;

- a HPV genotyping scheme for an accurate processing of results

[37] In a preferred embodiment of the invention, the above-mentioned kit also comprises a standard nucleic acid amplification mix with a pair of primers to test DNA integrity.

[38] In other preferred embodiment of the invention the said kit may also comprise a positive and negative control sample.

[39] Finally, the kit also may include instructions for its use.

[40] Examples EXAMPLE 1 : DNA extraction from cells of the cervix uteri - Cervical cells collected from the cervix uteri, using a brush or spatula, were resuspended in PreservCyt solution (Cytyc Corporation. Boxborough, MA. USA). An aliquot of 5ml was cen- trifuged at 10.000 rpm for 15 min. The pelleted cells were lysed in a Tris-buffered solution (0.3M NaCl, 1OmM EDTA, 10mM Tris pH 7.5), containing 0.5% SDS and 0.5μg/μl of proteinase K, at 56 ºC. The DNA was purified by standard phenol/ chloroform extraction and ethanol precipitation.

[41] EXAMPLE 2: Human papillomavirus detection - HPV DNA was detected by PCR using the degenerate consensus primers MY09/11 (Manos et al. Cold Spring Harbor Press. 1989, 7:209-214), which amplify a region of 449-458 nucleotides (depending on HPV type) of the highly conserved Ll ORF. Each PCR amplification was carried out with lOOng of template DNA in 1x PCR Buffer (Biotools, Madrid, Spain), containing 1.5mM MgC12, 12.5 pmol of each primer MY09 and MYl 1 (SEQ ID NO 1 and 2), 200 uM of each deoxynucleotide triphosphate and 1 unit of Taq DNA polymerase (Biotools, Madrid, Spain). Thermal cycling was performed in a Perkin-Elmer 9600 (PerkinElmer, MA, USA) as follows: denaturation of DNA template at 95 ºC for 5 min, followed by 45 cycles of 94 ºC for 60 sec, 55 ºC for 60 sec, and 72 ºC for 60 sec, and a final extension step at 72 ºC for 8 min. To check the integrity of the DNA extracted from the specimens, a region of 501 base pairs of the cellular Beta-globin gene was amplified using primers Betal and Beta2 (SEQ ID NO 21 and 22). The PCR conditions were the same as described for HPV detection. All PCR reactions were carried out under conditions that minimize sample cross-contamination. DNA from HeLa and CaSki cell lines was used as positive control. A negative control containing all PCR reagents, except the DNA, was added to monitor contamination. In order to verify the amplification efficiency of PCR reactions and the absence of unspecific products, the amplified DNA was analyzed in a horizontal 2% agarose gel, pre-stained with Gel Star (Cambrex Bio Science Rockland, Inc., ME, USA) and visualized on a UV screen.

[42] EXAMPLE 3: Human papillomavirus genotyping - MY09/11-positive cases were typed by RFLP analysis in 4 independent reactions. Each restriction reaction was performed in a final volume of 20 μl, using 1 μg of MY09/11 PCR product, 2 μl of 10x recommended restriction buffer, and 10 units of the following restriction en- donucleases: Pstl (Qbiogene, CA, USA), HaeIII (Fermentas Inc., Canada), Ddel (Qbiogene, CA, USA) and Rsal (Fermentas Inc., Canada), according to the manufacturers' instructions. Reactions took place at 37 ºC for 1 h. Digested products were electrophoretically separated on 10% polyacrylamide gels, in the presence of 25 bp and 50 bp DNA molecular weight markers (Invitrogen, Carlsbad, CA, USA). Gels were stained with Gelstar (Cambrex Bio Science Rockland, Inc., ME, USA) for 15 min and patterns were visualized using a Versa Doc Imaging System (Bio-Rad. Hercules. CA, USA). The identification of HPV type(s) was performed using a genotyping scheme (Figure 2).

[43] EXAMPLE 4: Validation of RFLP restriction reaction - To test the efficacy of each enzyme and to avoid the misidentification of types due to partial digestion, an additional restriction reaction was performed, in which MY09/11 PCR products and a control DNA (PCR product of Beta-globin fragment amplification) were digested together. Each restriction reaction was performed in a final volume of 20 μl, using 1 μg of MY09/11 PCR product. 1 μg of Betal/2 amplicon, 2 μl of 10x recommended restriction buffer, and 20 units of each restriction endonuclease, according to the conditions described in Example 3.

[44] EXAMPLE 5: Clinical validation of the present invention - To confirm the reliability of the present PCR-RFLP based methodology and the applicability of the HPV typing scheme in HPV identification, two different experiments were performed. The first study was carried out, in a blind fashion, on pre-selected HPV positive clinical samples, in which 27 different mucosal HPV types had been previously identified by PCR with MY09/11 primers and DNA sequencing (HPV 6, 11, 16, 18, 31, 33, 34, 35, 39, 44, 45, 52, 53, 56, 58, 59, 61, 62, 66, 67, 70, 71, 73, 81, 83, 84, and 102). In all samples, PCR-RFLP data consistently matched the DNA sequencing results. Figure 4 shows the RFLP patterns of some HPV types (HPV 16, HPV 58 and HPV 102).

[45] The second study, which has consisted in a random and blind experiment, was performed on a representative collection of 30 untyped clinical samples (10 normal samples, 10 low-grade squamous intraepithelial lesions, 5 high-grade squamous intraepithelial lesions and 5 invasive cervical carcinoma samples). Of the 30 cervical samples analyzed, HPV DNA was detected in 16 cases (3/10 normal samples, 3/10 low grade-lesions, 5/5 high grade-lesions and 5/5 carcinoma samples). Corresponding MY09/11 PCR products were subsequently genotyped using both the present invention and DNA sequencing/cloning technique. In the last, PCR products were purified from agarose gel using a PCR clean-up Gel extraction Kit (NucleoSpin® Extract II, Macherey-Nagel, Geπnany) and cloned. At least, five fragments of each amplicon were sequenced. Forward and reverse sequencing reactions were carried out using 20 ng of template DNA from PCR amplimers and 20 μM of the MY09 and MYl 1 primers, respectively. Automated DNA sequencing was performed by Eurofins MWG Operon (Ebersberg, Germany). Sequences obtained were compared with all sequences available in Gen-Bank database. HPV type was identified based on >98% sequence homology over 449-458 nucleotides. Table 2 shows the comparative genotyping results of HPV-positive clinical samples using RFLP analysis and DNA sequencing/ cloning analysis of MY09/1 1 PCR products. [46] Table 2:

[47] In Table 2, accession numbers of DNA sequences are given in brackets. Letter S means single infection and M means multiple infections.

[48] RFLP data consistently matched the DNA sequencing results, with the exception of one single case, sample 22, in which it was impossible to discriminate the triple HPV infection using RFLP analysis, due to the complexity of the pattern obtained. Nevertheless, in all other HPV-positive samples, even in the case of multiple infections (e.g. samples 16 and 23), sequencing results confirmed RFLP genotyping. AU the enzymes have shown a high cutting efficiency and no partial digestions were visualized under the RFLP conditions previously described.

Claims

[1] An assay for detection and individual identification of human papillomavirus (HPV) in a sample, which comprises the steps of: a) amplifying the DNA using HPV universal primers; b) analyzing the amplified fragment in independent restriction reactions; and c) identifying HPV type by comparison of the RFLP patterns with the reference patterns of known genotypes, characterized by step b) being conducted in 4 independent reactions with different restriction enzymes, namely Pstl, Haelll, Ddel and Rsal, and step c) being performed with the genotyping scheme of figure 2.

[2] The assay, according to claim 1. wherein the restriction products are analyzed in agarose or polyacrylamide gels pre-stained with a nucleic stain for DNA, being the concentration of the agarose at least of 3% and the concentration of polyacrylamide at least 10% in the respective gels.

[3] The assay, according to claims 1 or 2, wherein restriction products are analyzed in polyacrylamide gels pre-stained with a nucleic stain for DNA, wherein the concentration of said polyacrylamide gels is higher than 10% and up to 20%.

[4] The assay, according to any of claims 1 to 3, further comprising an amplification of human DNA in a sample.

[5] The assay, according to claim 4 wherein the sample of the DNA amplified was collected from the same origin as the tested sample.

[6] The assay, according to any of claims 1 to 5 further comprising a parallel co-digestion of the HPV DNA amplification products with the control DNA.

[7] A kit for performing the method of any of the claims 1 to 6, comprising the following components:

- a standard nucleic acid amplification mix with universal HPV primers;

- 4 different restriction reaction vessels each one comprising a specific restriction enzyme and respective buffers;

- a HPV genotyping scheme.

[8] A kit, according to claim 7, further comprising a standard nucleic acid amplification mix with a pair of primers to test DNA integrity.

[9] A kit, according to any of claims 7 or 8, further comprising the positive and negative control samples.

[10] A genotyping scheme according to figure 2.

[11] A genotyping scheme according to claim 10, wherein the HPV type is identified through the below described steps: - first, following the dashed or draw lines, according to the occurrence or non- occurrence of Pstl. HaeIIL Ddel and Rsal restriction reactions, respectively.

- secondly, by comparison of the restriction fragments obtained by Rsal digestion with the expected sizes defined for each HPV type, at the end of the scheme,

- in cases marked with caps letters, HPV type is identified by comparison of the restriction fragment sizes obtained by Ddel or HaeIII restriction digestⁱon