EP1124993A2 - Nouvelles amorces et sondes destinees a la detection du vih - Google Patents

Nouvelles amorces et sondes destinees a la detection du vih

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Publication number
EP1124993A2
EP1124993A2 EP99969965A EP99969965A EP1124993A2 EP 1124993 A2 EP1124993 A2 EP 1124993A2 EP 99969965 A EP99969965 A EP 99969965A EP 99969965 A EP99969965 A EP 99969965A EP 1124993 A2 EP1124993 A2 EP 1124993A2
Authority
EP
European Patent Office
Prior art keywords
hiv
oligonucleotides
subtypes
oligonucleotide
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP99969965A
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German (de)
English (en)
Inventor
Gerd Haberhausen
Pia Kasper
Christoph Kessler
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Roche Diagnostics GmbH
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Roche Diagnostics GmbH
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Filing date
Publication date
Application filed by Roche Diagnostics GmbH filed Critical Roche Diagnostics GmbH
Publication of EP1124993A2 publication Critical patent/EP1124993A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS

Definitions

  • the invention relates to a method for the subtype and / or cross-species detection of HI viruses in a sample as well as suitable oligonucleotides.
  • HIV is of great importance in analytical diagnostics.
  • detection methods which are based on the immunological detection of HIV-specific antibodies, HIV-specific proteins, for example reverse transcriptase, or of HIV-specific nucleic acids.
  • PCR polymerase chain reaction
  • PCR or polymerase chain reaction allows the amplification of nucleic acid sections with the help of oligonucleotides, so-called primers, which hybridize specifically with the nucleic acids to be detected. This results in amplification products, which in turn can be detected with further specific oligonucleotides, so-called probes.
  • a prerequisite for a successful PCR for the detection of HIV is, on the one hand, the most exact possible match of the complementary base sequence of the primers used with that of the nucleic acid to be detected so that the hybridization is as specific as possible. On the other hand, however, it is also advantageous to be able to amplify as many variants of HIV as possible with the same primers.
  • subtypes The different types of HIV-1 are commonly referred to as subtypes. At least 9 subtypes are currently known, which are referred to as subtypes A to H and O (Human Retroviruses and AIDS, Los Alamos, Natl. Laboratory, Los Alamos, New Mexico, 1994; editor G. Meyers et al., IA-1) . So far no primers or probes have been developed with which all known subtypes of HIV-1 can be recognized. It would also be advantageous to also be able to recognize HIV-2 and its subtypes with the same primers and probes. Subtypes A, B, C and D are currently known from HIV-2.
  • WO96 / 02557 describes general uses of oligonucleotides to inhibit HIV propagation and to detect the virus. However, there are no indications of suitability for cross-species or cross-type detection.
  • primers and probes are described, each with base sequences from conserved regions of the gag, vpr and po / genes of the HIV-1 isolates Bru, Mal and Eli and hybridize with the corresponding regions of HIV-2 ROD and SIV Mac. Furthermore, primers and probes are disclosed, each of which hybridizes with sections from the env, nef ⁇ , vif and vpr genes of HIV-1 Bru, Mal and Eli, and those which with sections from the nefl, vifl and Hybridize vpx genes from HIV-2 ROD and SIV Mac. Although some of these oligonucleotides apparently hybridize across species, nothing is said about which subtypes of the individual viruses are recognized.
  • EP 839 917 in which, however, only subtypes of HIV-1 can be detected
  • EP 887 427, WO99 / 07898 and WO98 / 58086 discloses primers and probes which amplify a sequence section from the o / gene of HIV-1 and can thereby recognize five subtypes of HIV-1.
  • EP 0 617 132 also discloses primers and probes for the detection of HIV-1 which are able to differentiate between HIV-1 and its phylogenetically closest relatives, such as HTLV-II or HIV-2.
  • the oligonucleotides selected there hybridize with a number of regions from the HIV genome, including the LTR and most structural genes. However, there is no evidence of suitability for cross-subtype detection.
  • oligonucleotides for purification by means of hybridization with target oligos, e.g. WO98 / 27425.
  • target oligos e.g. WO98 / 27425.
  • oligos are not suitable for cross-subtype detection of HIV.
  • WO90 / 01069 discloses oligonucleotide pairs for the detection of HIV, these pairs are not used as primers for an enzymatic Amplification used, but they hybridize on the same strand and represent the sequence sought even after their linkage.
  • the object of the present invention was therefore to provide a method for the subtype and / or cross-species detection of HI viruses in a sample.
  • This object is achieved according to the invention by a method for the detection of nucleic acids of HI viruses in a sample across and between species
  • This object is preferably achieved by a method for the subtype and / or cross-species detection of nucleic acids of HI viruses in a sample Hybridization of nucleic acids with one
  • Oligonucleotide combination comprising two or more oligonucleotides hybridizing specifically with HIV nucleic acids, each of at least 10 consecutive nucleotides from (i) a highly conserved region of the LTR region, the gra ⁇ gene or the p ⁇ gene of HIV, represented by one of those in SEQ
  • ID NO: 1 to 13 specified sequences, (ii) a corresponding region of another HI virus isolate, (iii) a corresponding region of a consensus sequence derived from a number of HI virus isolates, or sequences complementary thereto, and carrying out an amplification step.
  • the method according to the invention enables the detection of more subtypes of HIV-1 and HIV-2 (cross-subtype detection) than was possible in the prior art.
  • Cross-subtype detection means that at least 2 subtypes of a particular species can be detected with a single probe or a single oligonucleotide combination.
  • the method according to the invention now makes it possible to recognize at least 7 of 9 subtypes of HIV-1, in particular including subtype O in conjunction with other subtypes, with the aid of particularly fewer and in the best case with the same oligonucleotides.
  • all 9 subtypes known so far can be recognized.
  • At least 2, preferably at least 3 and most preferably, all subtypes A, B, C and D of HIV-2 can be identified by the cross-subtype detection of HIV-2.
  • nucleic acids from HIV-1 and HIV-2 can also be detected across species.
  • Cross-species means that different species of immunodeficiency viruses are recognized with the same oligonucleotides, e.g. HIV-1 and HIV-2.
  • At least 7 of the currently known 9 subtypes of HIV-1 and further of the currently known subtypes of HIV-2 are preferably detected.
  • all 9 subtypes of HIV-1 plus further subtypes of HIV-2, particularly preferably plus all currently known subtypes of HIV-2 can be detected.
  • the method according to the invention is based on an amplification of nucleic acid sections of HI viruses with the aid of specific oligonucleotides which can act as primers or as probes.
  • oligonucleotide is a single-stranded linear nucleic acid molecule.
  • oligonucleotides have 10 to 100 bases.
  • the oligonucleotides according to the invention are preferably 10 to 80, particularly preferably 10 to 60, even more preferably 10 to 30 and most preferably 20 to 30 nucleotides long.
  • nucleic acids a distinction is made between oligodeoxyribonucleotides and oligoribonucleotides, but oligoribonucleotides also include compounds in which the hydrogen of the hydroxyl group is replaced by organic radicals, for example an allyl group. Such connections have long been known to the person skilled in the art.
  • oligonucleotide may also include, for example, molecules in which the sugar phosphate backbone is replaced by a peptide backbone. This group of compounds is called PNA. All oligonucleotides have in common that they have bases on the backbone which are capable of hydrogen bonds with bases which are complementary thereto.
  • the bases include the natural bases A, G, C, T and U, but also artificial bases, such as Deaza-G.
  • An oligonucleotide primer is an oligonucleotide which can hybridize with a second, likewise single-stranded nucleic acid and subsequently can be supplemented with a suitable enzyme, for example a DNA polymerase along the second nucleic acid serving as template with nucleoside triphosphates, so that a double-stranded nucleic acid is formed.
  • a suitable enzyme for example a DNA polymerase along the second nucleic acid serving as template with nucleoside triphosphates, so that a double-stranded nucleic acid is formed.
  • the primer has a hydroxyl group at its 3 'end, ie at the end at which nucleotide building blocks are attached, at the 3' C atom of the sugar.
  • An oligonucleotide combination comprises several oligonucleotides, preferably it comprises a pair of primers or a combination of primers and probes, such as a pair of primers and a probe.
  • a pair of primers consists of two oligonucleotide primers, which allow the preferential enzymatic amplification of a certain section of a nucleic acid.
  • the two primers of the pair of primers preferably hybridize with different strands of the original nucleic acid in such a way that the respective extension products overlap one another. This then leads to the fact that each of the primers can hybridize with the extension product of the respective other primer and a section which lies between the two primers is multiplied as the amplification product.
  • probes are also single-stranded oligonucleotides, which can also act as primers, but are primarily intended to specifically hybridize with already amplified nucleic acid sections in order to enable nucleic acid detection.
  • oligonucleotide thus includes the terms primer and probe, which differ only in their function.
  • the oligonucleotides used in the method according to the invention can also contain labeling groups, such as radioactive labels or fluorescent labels. In particular, it is the probes that have markings.
  • Amplification is understood to mean the duplication of nucleic acids or nucleic acid sections.
  • a well-known amplification method is the PCR mentioned at the beginning. In this method, a normally double-stranded DNA molecule is first denatured, ie into its Single strands split. The amplification primers are then added under conditions that allow the primers to hybridize with the target DNA sequence. The primers are then supplemented along the nucleic acid matrix with the aid of a polymerase enzyme and nucleotide building blocks. The newly formed double-stranded nucleic acids are then denatured again and a new polymerase cycle begins. Conventional PCR methods typically use between 25 and 40 cycles. An amplification in the sense of the invention can therefore also include necessary preparatory steps for nucleic acid amplification, such as denaturing the double-stranded DNA.
  • hybridization here means the combination of two complementary single nucleic acid strands into a double strand.
  • the single strands do not have to be 100% complementary, but can have deviations in the base sequence.
  • the single strands in this case the oligonucleotides, must be sufficiently specific under the prevailing hybridization conditions.
  • the oligonucleotides used must meet at least two conditions. On the one hand, they have to be specific enough to hybridize exclusively with nucleic acids derived from HI viruses. On the other hand, these viruses vary greatly in some genome regions, ie there are relatively large differences in the base sequence. Because of these differences, a distinction is made between HIV-1 and HIV-2 as well as between so-called subtypes, which are relatively closely related strains of the same virus.
  • oligonucleotides of the present invention In relation to the oligonucleotides of the present invention, this means that they must also be able to recognize not only a specific virus or a specific subtype, but they must have a base sequence that hybridizes with as many or as possible allowed even all known subtypes of HIV-1 or HIV-2 or even both.
  • oligonucleotides For this purpose, one normally selects the oligonucleotides from relatively conserved regions of the genome of the viruses to be detected and then uses the respective complementary sequence. From the state of the
  • oligonucleotides have now been found in the present invention which enable both cross-type detection and cross-species detection of HIV. These oligonucleotides hybridize to newly discovered, highly conserved regions of HIV. These are located in the LTR region, the gag and po / genes. These regions are relatively small, but with an average length of 50 to 100 nucleotides they allow hybridization with one or more oligonucleotides. The determination of the regions of the HIV genome is based here, as in most of the cited prior art documents, on the numbering of the HIV-1 isolate HXB2, as published in: Wong-Staal et al., Nature 313, 277- 284 (1985).
  • sequences of these new highly conserved areas are in SEQ ID NO. 1 to 13 indicated, the sequences of which refer to the sequence of HIV-1 HXB2, accession number K03455 from the HIV Sequence Database (http: //HIV-web.lanl. Gov./).
  • the location of the highly conserved sequences is as follows:
  • SEQ ID NO. 1 LTR region, position 504-565, length 62 nucleotides
  • SEQ ID NO. 2 gag gene, position 761-822, length 62 nucleotides
  • SEQ ID NO. 3 gag gene, position 1786-1847, length 62 nucleotides
  • SEQ ID NO. 4 po / region, position 2307-2360, length 54 nucleotides
  • SEQ ID NO. 5 pol gene, position 2376-2434, length 59 nucleotides
  • SEQ ID NO. 6 po / gene, position 2568-2632, length 65 nucleotides
  • SEQ ID NO. 7 po / gene, position 3093-3145, length 53 nucleotides
  • SEQ ID NO. 8 pol gene, position 4131-4207, length 77 nucleotides
  • SEQ ID NO. 9 po / gene, position 4333-4399, length 67 nucleotides
  • SEQ ID NO. 10 pol gene, position 4638-4696, length 59 nucleotides
  • SEQ ID NO. 1 1 p ⁇ / gene, position 4884-4984, length 101 nucleotides
  • SEQ ID NO. 12 po / gene, position 5034-5095, length 62 nucleotides
  • SEQ ID NO. 13 pol gene, position 4410-4506, length 97 nucleotides.
  • oligonucleotides suitable according to the invention preferably have base sequences which lie within the highly conserved regions mentioned above or their complementary sequences.
  • overlap is to be understood here so that the oligonucleotides according to the invention each overlap with at least 10 successive bases from one of the highly conserved regions.
  • the oligonucleotides preferably overlap with one of the regions with SEQ ID NO: 4, 5, 9, 1 0 and 1 3, preferably 4, 5, 9 and 10.
  • the oligonucleotides each overlap with one of the regions SEQ ID NO: 6, 8, 10, 1 1 and 1 3.
  • Oligonucleotide sequences suitable according to the invention can also have a base sequence which is a consensus sequence from highly conserved regions of several HIV virus isolates or strains. That that, for example, a base sequence of several bases corresponds to one HIV virus isolate and another base sequence in the same oligonucleotide corresponds to another HIV virus isolate.
  • the oligonucleotide then contains heterologous base sequences.
  • the oligonucleotides preferably each comprise at least 10 consecutive nucleotides from one of the regions mentioned above. More preferably, they contain 15 to 30 such nucleotides.
  • the method according to the invention comprises at least the following steps:
  • an amplification step is preferably carried out.
  • the subtype and / or cross-species detection of HIV viral nucleic acids is made possible by using at least two oligonucleotides according to the invention.
  • These oligonucleotides are preferably used as amplification primers in such a way that they hybridize, for example, in each case close to one end of one of the highly conserved regions and thus produce an amplification product (preferably PCR) which corresponds to a section of the highly conserved region in question.
  • This amplification product and thus the HIV-specific nucleic acid can then be detected with the aid of a probe which is either one of the oligonucleotides used as primer or an additional oligonucleotide which hybridizes within the amplified sequence.
  • a probe which is either one of the oligonucleotides used as primer or an additional oligonucleotide which hybridizes within the amplified sequence.
  • DNA-binding reagents such as a DNA-binding dye (e.g. Sybergreen) can also be used in the detection (WO97 / 46707).
  • oligonucleotide combinations or primer pairs are used, each of which only one oligonucleotide primer lies within one of the conserved regions, while the other primer lies outside, so that the amplification product thus produced only partially from a base sequence from one of the highly conserved ones Regions.
  • the latter primer is preferably subtype-specific and / or species-specific, so that subtypes or the species can be specifically detected with such a combination.
  • two or more pairs of primers are preferably used as the oligonucleotide combination, at least two oligonucleotides each containing at least 10 consecutive nucleotides from a sequence (i), (ii), (iii) or sequences complementary thereto, as described above.
  • the entirety of the primer combinations thus allows sub-type and / or cross-species detection of HI viruses.
  • an oligonucleotide combination suitable for the method according to the invention comprises at least two, preferably three oligonucleotides (e.g. a primer pair or a primer pair plus probe), each of which consists of at least 10 nucleotides
  • (iii) contain a corresponding region of a consensus sequence derived from a number of HIV virus isolates, or sequences complementary thereto.
  • the at least one and preferably the at least two oligonucleotide (s) are preferably selected so that they enable cross-subtype detection of HIV-1, i.e. that nucleic acids of at least 7 of subtypes A, B, C, D, E, F, G, H and O of HIV-1, preferably of all subtypes, are recognized.
  • At least one and preferably at least two oligonucleotide (s) are used for cross-subtype detection, each of which contains at least 10 consecutive nucleotides from (i) a highly conserved region of the LTR, gag gene or pol gene of HIV, represented by one of the in SEQ ID NO: 1, 2,
  • oligonucleotide For sub-type and / or cross-species detection of HIV-1 and HIV-2, preference is given to using at least one and preferably at least two oligonucleotide (s) which each comprise at least 10 consecutive nucleotides
  • oligonucleotides are particularly suitable for the method according to the invention. These oligonucleotides are shown in the sequences given in SEQ ID NO: 14 to 25. As already mentioned at the beginning, these oligonucleotides can consist of natural or synthetic nucleic acid building blocks, or even the PNA already mentioned. At least one of the oligonucleotides used in the method according to the invention preferably carries one or more labels. Fluorescence markers or radioactive markers, such as [ 32 P] -labelled nucleotides, are suitable as labels.
  • Another object of the invention is an oligonucleotide which contains at least 10 consecutive nucleotides from (i) a highly conserved region of the po / gene of HIV, represented by one of those given in SEQ ID NO: 4, 5, 9 or 10
  • the oligonucleotide according to the invention preferably comprises or is one of those shown in SEQ ID NO. 14 to 25 oligonucleotides shown.
  • the length of the oligonucleotide according to the invention is preferably 10 to 80 nucleotides. It particularly preferably comprises approximately 20 to 30 bases and has a GC content of between 40 and 60%. It is also advantageous if the oligonucleotides have no self-complementarity at the 3 'end and, moreover, do not have a CG run at the 3' end.
  • a GC run is a base sequence that consists predominantly or exclusively of bases C and G. Furthermore, the oligonucleotides should not contain any palindromes.
  • the oligonucleotides according to the invention preferably have a maximum of 2 mismatches with the corresponding sequences of the same positions of all subtypes which hybridize with them.
  • An oligonucleotide according to the invention preferably has no mismatches with nucleotide acids of subtypes A, B, C, D, E, F, G, H and O of HIV-1 and / or of subtypes A, B, C and C at its 3 'end from HIV-2 to.
  • the primers are chosen such that the 5 'ends of the primers are positioned at a maximum of 80 bases from one another, based on the regions in which the primers hybridize to the nucleic acid to be detected.
  • Particularly preferred primer pairs are those in which a further HIV-specific sequence lies within this range that can be amplified by the primers. This sequence can then preferably be used to detect the amplification products with the aid of a probe specific for it.
  • the oligonucleotides according to the invention are preferably provided with at least one of the labeling groups mentioned above.
  • a further aspect of the present invention is thus also a combination of two or more oligonucleotides, both of which have the above-mentioned properties according to the invention and which in their entirety are suitable for the detection of HIV viruses across subtypes and / or species.
  • the invention further comprises combinations of oligonucleotides. Combinations of at least two oligonucleotides are preferred, each comprising at least 10 consecutive nucleotides from (i) the same highly conserved region of the LTR region, the gag
  • the gene or the po / gene of HIV represented by one of the sequences given in SEQ ID NO: 1 to 1 3, (ii) a corresponding region of another HIV virus isolate, (iii) a corresponding region of one of several HIV virus isolates derived consensus sequence, or sequences complementary thereto.
  • Yet another aspect of the present invention is a combination of several oligonucleotides, at least two oligonucleotides according to the invention being present, as well as further oligonucleotides, each of which contains a sequence specific for a single subtype of HIV-1 and / or HIV-2, the total of the Oligonucleotides in the oligonucleotide combination allow sub-type and / or cross-species detection of HI viruses.
  • a further object of the present invention is to provide a reagent kit which contains at least one oligonucleotide according to the invention or an oligonucleotide combination according to the invention as a primer or as probes for the detection of HI viruses or their nucleic acids, and for carrying out a hybridization and amplification of nucleic acids in a sample includes.
  • the invention relates to the use of oligonucleotides or oligonucleotide combinations as primers and / or probes for the detection of HI viruses, in particular for the detection of subtypes and / or species.
  • Sample preparation First 420 ⁇ l plasma was mixed with 80 ⁇ l Proteinase K (25 mg / ml) and vortexed for a few seconds. Then 500 ⁇ l lysis buffer (5.4 M guanidinium thiocyanate, 10 mM urea, 10 mM Tris-HCl, 20% Triton X100, pH 4.4) were added, which contained 1 ⁇ g carrier RNA (polyA / ml). The mixture was vortexed and then shaken for 10 minutes at room temperature. Then 500 ⁇ l of isopropanol-MGP were added (6 mg of magnetic glass particles in isopropanol). The mixture was vortexed again and then for 20 minutes Shaken at room temperature.
  • lysis buffer 5.4 M guanidinium thiocyanate, 10 mM urea, 10 mM Tris-HCl, 20% Triton X100, pH 4.4
  • the MGPs were separated from the solution by magnetic separation. The supernatant was removed and discarded. 750 ul wash buffer (20mM NaCl, 20mM Tris-HCl, pH 7.5, 70% ethanol) was added, the MGPs were resuspended by vortexing, and magnetic separation was performed again. The washing process was repeated 5 times in total and finally 100 ⁇ l of DEMC water was added for the elution. The mixture was shaken for 15 minutes at 80 ° C. and then a further magnetic separation was carried out. 10 ⁇ l of the eluate were used for an RT-PCR.
  • Table 1 shows the primers and probes used, as well as their associated highly conserved region, position in the genome and the amplification product produced with primer pairs.
  • This primer sn ewe s pu certe rmer un on en by ⁇ c e are new primers from the po / region of the HIV genome in accordance with the highly conserved areas listed in Table 1.
  • Amplification mix and thermal cycler protocol for the RT-PCR Master mix: Final reagent concentration in the master mix
  • the entire detection reaction was fully automated using an Elecsys ® 1010 automated analyzer.
  • the signals of the new primers show a similar sensitivity to the references. There is a very good signal gradation within the dilution series.

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Abstract

L'invention concerne un procédé de détection de sous-types et d'espèces du virus de l'immunodéficience humaine dans un prélèvement à l'aide d'au moins un oligonucléotide contenant au moins 10 nucléotides consécutifs comprenant (i) une région à haute conservation de la région LTR, du gène-gag ou du gène-pol de VIH, (ii) d'une région correspondante d'un autre isolat de VIH, (iii) d'une région correspondante d'une séquence consensus composée de plusieurs isolats de VIH, ou de séquences complémentaires.
EP99969965A 1998-10-30 1999-10-29 Nouvelles amorces et sondes destinees a la detection du vih Withdrawn EP1124993A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19850186 1998-10-30
DE1998150186 DE19850186A1 (de) 1998-10-30 1998-10-30 Neue Primer und Sonden zum Nachweis von HIV
PCT/EP1999/008211 WO2000029611A2 (fr) 1998-10-30 1999-10-29 Nouvelles amorces et sondes destinees a la detection du vih

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JP (1) JP2004500014A (fr)
DE (1) DE19850186A1 (fr)
WO (1) WO2000029611A2 (fr)

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JP6593984B2 (ja) * 2014-09-26 2019-10-23 株式会社Ｋｒｉ Ｒｎａ増幅法およびｈｉｖ−１感染診断方法
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JP6858008B2 (ja) * 2016-11-30 2021-04-14 シスメックス株式会社 粒子分散装置及び粒子分散方法
CN109576397B (zh) * 2018-12-28 2022-10-14 广州达安基因股份有限公司 一种人类免疫缺陷病毒1型核酸定量检测试剂盒
CN116064922A (zh) * 2021-10-29 2023-05-05 京东方科技集团股份有限公司 一种人类免疫缺陷病毒1型核酸测定试剂盒及其操作方法

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WO2000029611A2 (fr) 2000-05-25
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JP2004500014A (ja) 2004-01-08
DE19850186A1 (de) 2000-05-25

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