EP1220951A2 - Essai de transcriptase inverse - Google Patents

Essai de transcriptase inverse

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Publication number
EP1220951A2
EP1220951A2 EP00966304A EP00966304A EP1220951A2 EP 1220951 A2 EP1220951 A2 EP 1220951A2 EP 00966304 A EP00966304 A EP 00966304A EP 00966304 A EP00966304 A EP 00966304A EP 1220951 A2 EP1220951 A2 EP 1220951A2
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EP
European Patent Office
Prior art keywords
dna
activity
reverse transcriptase
retrovirus
rna template
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EP00966304A
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German (de)
English (en)
Inventor
Archibald Lovatt
Iain Doherty
Kenneth Thomas Smith
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Quip Technology Ltd
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Quip Technology Ltd
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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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • 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

Definitions

  • the present invention relates to a method for the detection of reverse transcriptase (RT) activity, and to a method for the detection of retroviruses.
  • the invention further relates to kits for use in the detection of RT activity and of retroviruses.
  • the invention yet further relates to a method and kit for the detection of porcine endogenous retrovirus (PoERV) .
  • Retroviruses are important infectious agents in humans and animals, and are responsible for a large number of diseases. These agents may be passed between hosts by, among other means, transfer of biological material, for example, organ transplants, blood transfusions and the like.
  • a source of particular concern is xenotransplantation; that is, organs/tissues transplanted across species.
  • xenotransplantation that is, organs/tissues transplanted across species.
  • certain retroviruses may be able to cross the species barrier, and so cause zoonoses.
  • One such retrovirus which has been shown to be able to cross species is porcine endogenous retrovirus (PoERV) ; this is of particular concern for xenotransplantation, as pigs are a favoured source of suitable organs/tissues.
  • PoERV porcine endogenous retrovirus
  • RT activity is normally associated with the presence of retroviruses and detection of RT activity in harvest material, cell supernatants or viral vaccine preparations can be diagnostic for retroviral contamination.
  • conventional RT testing has been performed using synthetic oligonucleotides as templates for RT and estimations of contaminating DNA polymerases, however the test is not sensitive enough to detect low levels of RT activity (Robertson et al . , 1997; Maudru and Peden, 1998) .
  • Product enhanced reverse transcriptase (PERT) assays also termed Amp-RT or polymerase chain reaction-based reverse transcriptase (PBRT) assays (Maudru and Peden, 1997) , have been reported to be up to 10 ⁇ -fold more sensitive than conventional RT assays for detecting the presence of retroviruses (Silver et al . , 1993; Pyra et al . , 1994; Heneine et al . , 1995; Roberston et al . , 1997; Arnold et al . , 1998).
  • retroviruses Silver et al . , 1993; Pyra et al . , 1994; Heneine et al . , 1995; Roberston et al . , 1997; Arnold et al . , 1998).
  • PERT assays are RT-dependent PCR assays and therefore combine the broad specificity of conventional RT assays with the high sensitivity of PCR. As with conventional RT assays, they are utilised to detect the RT activity packaged into extracellular retrovirus particles. The assays involve converting an RNA template to cDNA and then amplifying the cDNA using product-specific primers. As no exogenous RT activity is added to the reaction, cDNA will only be generated if the sample itself contains RT activity. If RT activity is not present, product will not be detected.
  • Detection of the PCR product may be achieved by conventional means after the end of the amplification process.
  • real-time detection of levels of PCR product during amplification may be achieved using TaqMan® PCR technology with the detection of specific PCR product accomplished using for example the ABI Prism 7700 Sequence Detection System (PE Biosystems, Foster City, California) .
  • a specific fluorogenic oligonucleotide probe with both a reporter and quencher dye attached anneals specifically to the amplified product between the amplimers.
  • the detection reaction uses the 5 • -nuclease activity of AmpliTaq GoldTM DNA polymerase (PE Biosystems) to cleave the reporter dye from the probe, which results in an increased fluorescence when reverse transcriptase activity is present.
  • a major problem with PERT assays is the potential for misinterpretation of test results, leading to the conclusion that a sample contains retroviral RT. This arises from the sensitivity of the test, coupled with the ability of DNA polymerases, telomerases and other cellular enzymes to utilize RNA templates, thereby showing RT-like activity ( ugert et al . , 1996, Silver et al . , 1993, Boni et al . , 1996).
  • F-PERT high throughput fluorescent PERT
  • a method for the detection of reverse transcriptase (RT) activity in a test sample comprising the steps of: a) contacting the test sample with an RNA template and an oligonucleotide that is complementary to a portion of the RNA template under conditions which allow the oligonucleotide and the RNA template to anneal and such that a DNA strand will be synthesised by extension from the oligonucleotide if reverse transcriptase is present in the sample; b) subjecting the resulting mixture to a treatment whereby any DNase present in the mixture is substantially inactivated; c) amplifying the synthesised DNA by use of a DNA polymerase, under conditions whereby the amplified DNA may be detected by incorporation or release of a label; and d) detecting any amplified DNA by way of the incorporation or release of the label.
  • RT reverse transcriptase
  • reverse transcriptase activity refers to biological enzymatic activity which is capable of generating a DNA strand from a template RNA strand, in the presence of suitable nucleotides, cations and buffers and the like, and under appropriate temperature and pH conditions.
  • the method is used to detect reverse transcriptase activity of a retrovirus present in the sample.
  • the method may be used to detect any retrovirus, for example FeLV, HIV and in particular porcine endogenous retrovirus.
  • test sample may comprise any biological tissue or body fluid (for example, cells, serum, plasma, semen, urine, saliva, sputum, cerebrospinal fluid) , and may be processed in any suitable manner in order to prepare the sample for testing.
  • the test sample may be clarified by low speed centrifugation at 10,000g and filtered through a 0.45 micron filter to remove debris. After this the test sample may be tested directly after mixing in disruption buffer.
  • the sample may be centrifuged at approximately 100,000 g for 1 h to sediment any virus from a test sample to concentrate it or remove any soluble inhibitors of RT activity. The sample would still be disrupted by detergent disruption buffer.
  • a test sample may be tested directly- without clarification and concentration by centrifugation.
  • An induction step may be included prior to assaying the sample in order to stimulate retrovirus production.
  • “Amplifying” refers to any procedure capable of increasing the number of copies of a particular nucleic acid sequence in a sample. Typically this amplification will be performed by means of the polymerase chain reaction (PCR) , although it may be possible to use alternative techniques, for example, the ligation amplification reaction (LAR) , or the ligase-based amplification system (LAS) , and the like. In usual embodiments of the present invention, several rounds of amplification/detection steps will be performed. Moreover, the amplification step (c) and detection step (d) may occur concurrently, allowing real-time detection of the generation of amplified DNA.
  • PCR polymerase chain reaction
  • LAR ligation amplification reaction
  • LAS ligase-based amplification system
  • the assay is as sensitive as other recently published conventional PERT (C-PERT) assays (Maudru and Peden, 1997) , and at least IO 4 times more sensitive than conventional RT (C-RT) assays.
  • C-PERT conventional PERT
  • C-RT conventional RT
  • amplification of the synthesised DNA is carried out under conditions such that during amplification a probe comprising an oligonucleotide, possessing a reporter molecule and a suppressor molecule, anneals to a strand of the template nucleic acid and whereby nuclease activity of the DNA polymerase cleaves one of said suppressor molecule and said reporter molecule from the probe. Thereafter the non-suppressed reporter molecule may be detected.
  • the reporter molecule is a fluorescent molecule.
  • the reporter molecule is FAM
  • the suppressor molecule is TAMRA (6-carboxy-N, N, N' , N * -tetramethyl rhodamine) .
  • the complementary oligonucleotide bears FAM at its 5* end, and TAMRA at its 3' end.
  • This system is the basis of the TaqMan® technology.
  • Other suitable reporter and or suppressor systems may of course be used.
  • DABYL 3'quencher label
  • a variety of fluorophores are available for conjugation to the 5 ' oligo terminus or to bind directly to products of
  • dyes include fluorescein (6-FAM) ,
  • the TaqMan® technology from ABI represents the newest development in high throughput PCR. This system has aided the detection and quantitation of pathogen DNA and RNA (Morris et al . , 1996; Kennedy et al . , 1998; Higgins et al . , 1998; Everett et al . , 1999; Josefsson et al . , 1999), and the rapid identification of retroviral reverse transcriptase activity (Arnold et al . , 1998). The incorporation of this technology into a PERT assay will be of benefit in retrovirus discovery and biosafety testing.
  • the main advantage of the TaqMan® technology is the reduced labour and sample manipulation.
  • the assay is also quantitative and direct quantification of RT in samples can be carried out rapidly without the need of achieving target-to-competitor ratios. Additionally, real-time PCR reactions are also less sensitive than endpoint PCR reactions to the effects of inhibitors, presumably because measurements are taken during the exponential phase where reaction components are not limiting.
  • the previously described assay by Arnold et al (1998) displayed a high level of background fluorescence. Without wishing to be bound by theory it is thought that this may be due to DNase present in the sample processing the probe and releasing the reporter molecule. The present assay therefore employs a DNase inactivation step.
  • the DNase inactivation step is a high-temperature protein denaturation step. Conveniently the reaction is held at at least 90°C for at least 5 minutes; more preferably the reaction is held at at least 95°C for at least 10 minutes. Alternatively a DNase inactivator such as a proteinase may be added.
  • a DNase inactivator such as a proteinase may be added.
  • DNA polymerases and telomerases that can generate an RT- like polymerisation with RNA templates.
  • the known RNA template lacks telomerase target sequences.
  • the known RNA template may be derived from Brome Mosaic Virus (BMV) RNA, or bacteriophage MS2 RNA.
  • BMV Brome Mosaic Virus
  • suitable RNA templates may of course be used, for example, genomes of RNA viruses with no DNA intermediate such as influenza virus, tobacco mosaic virus or the bacteriophage of the Leviviridae .
  • any purified RNA would provide a suitable template for RT if an appropriate control reaction were included.
  • the sequence specificity of telomerases is such that there is no target template sequence in the BMV or MS2 fragment amplified and therefore, no potential for a signal from the RT-like activity associated with the enzyme (Blackburn 1993) .
  • the RT reaction may be carried out at a pH above 5.5, and preferably also at a pH of below 8.5 and preferably at a pH of about 8.2. However, the reaction may be carried out at a pH of 3 to 10 but is not efficient at the extremes of this pH range.
  • Several laboratories have employed methods that suppress the RT-activity associated with cellular DNA polymerases (Lugert et al . , 1996; Chang et al . , 1997). Chang et al . , 1997 have reported that authentic RT activity has a broader pH range than cellular polymerase RT-like activity. They have reported that lowering the pH of the RT reaction to pH 5.5 suppresses non-retroviral RT activity.
  • RT or RT-like activity was not detected in CHO and BHK-21 cell lysates at the lower pH of pH 5.5. This is of concern as endogenous retroviral particles and RT activity have been detected in association with CHO (Anderson et al . , 1991; Dinowitz et al . , 1992; Adamson, 1998) and BHK-21 (DeHaven et al . , 1998) cells (Table 6). Therefore, it is likely that lowering the pH of the RT reaction has detrimental affects on the detection of authentic RT activity of some retroviral types and may allow the misinterpretation of data and the reporting of false RT- negative test samples.
  • the known RNA template is at a concentration of less than 6 ng/ ⁇ l in the reaction mix. In a preferred protocol, the RNA concentration may be less than 1.5 ng/ ⁇ l in the reaction mix.
  • Cell lysates provide a source of cell-associated polymerases and the signal generated from such samples was not observed on reduction of the BMV RNA template level from 60 ng to 15 ng and below. However, the detection of RT activity was unaffected. This observation may reflect a reduced affinity for RNA templates of DNA polymerases. In addition, the generation of RT-like activity by DNA polymerases was less reproducible than the genuine retroviral RT activity observed.
  • the method step a) includes a suppressor of RT activity of DNA polymerases.
  • the suppressor may be activated DNA.
  • the suppressor may be activated calf thymus DNA (aCT DNA) .
  • the suppressor may be included in the reaction mix in excess of the known RNA template; in the case of aCT DNA, this is for example a ratio of at least 1:26 template: aCT; or more preferably at least 1:104, and most preferably at least 1:416.
  • the concentration of aCT DNA in the RT reaction mix is at least 150 ng/ ⁇ l.
  • Activated calf thymus DNA has previously been observed to essentially suppress the positive signals produced by DNA polymerases with no reduction in RT activity (Lugert et al . , 1996).
  • aCT Activated calf thymus DNA
  • kits for use in the detection of reverse transcriptase (RT) activity in a sample including a combination of the necessary reaction reagents, oligonucleotides, RNA templates activated DNA and the like for use in the methods outlined above.
  • a kit as described above for use in the detection of porcine endogenous retrovirus (PoERV) in a test sample may include a porcine endogenous retrovirus specific RNA template. Details of suitable PoERV specific RNA templates may be found in co- pending patent applications WO97/40167, W097/21836 and GB9919604.0.
  • the PoERV RNA template may be synthesised in vitro and may for example correspond to positions encompassing the positions 805 to 902 of the PoERV genome (see Figure 3 of WO97/40167) .
  • the PoERV-grag-specific oligonucleotides used would be derived from sequences which correspond to approximate positions 805-823 (Forward) , to position 828 to 854 (Probe) and position 902-877 (Reverse) on the PoERV genome (see
  • oligonucleotide primers used for this amplification would be as follows:
  • the fluorescent 5' -FAM (6-carboxy fluoresceine) and 3' -TAMRA (6-carboxy-N, N, N' ,N' -tetramethylrhodamine) quencher labelled probe would be as follows:
  • Probe 5 • -CCCTATATCCTTACGTGGCAAGATTTG-3 ' .
  • the natural primer glycine 2 tRNA
  • the PBS primer binding site
  • U5 region of the LTR long terminal repeat
  • Figure 1 shows (a) the relationship between C ⁇
  • FIG. 2 shows a list of cell lines and virus stocks used in the following examples; Abbreviations: ATCC
  • Figure 3 shows detection limits using the presently described F-PERT assay for both retroviral particles and purified RT enzyme;
  • A retroviral particles
  • B purified
  • Figure 4 shows a comparison of the sensitivity of retroviral RT activities for both the present F-PERT assay and conventional RT assays; The detection limit is expressed as the highest dilution detected using each assay.
  • Figure 5 shows the effect of divalent cation concentration on RT activity;
  • Figure 6 shows the suppression of RT-like DNA polymerase activity by aCT DNA
  • aCT RT-like activity
  • B Amplification results obtained from 1-5 unit (U) of enzyme which gave the highest activity in F-PERT reactions without aCT DNA and suppression of that activity by aCT DNA (BMV: aCT ratio 1:104).
  • Taq (5U native Taq DNA polymerase).
  • oc (1U calf thymus DNA polymerase ⁇ ) .
  • ⁇ (1U human DNA polymerase ⁇ ) .
  • TdT 1U terminal deoxynucleotide transferase.
  • FIG. 7 shows the RT activity of various cell line supernatants, as tested by the present method.
  • Cell culture supernatants were tested with and without activated calf thymus DNA as indicated. IDU-induced cultures are identified by superscript a . Abbreviations; (+) positive and (-) negative for RT-like activity by C ⁇ ⁇ 32 or > 32 respectively.
  • Supernatants spiked with IO 6 rVLPs of a RT positive stock are shown as superscript b .
  • Those labelled as superscript c were spiked with 10 s rVLPs. All other supernatants were spiked with IO 3 rVLPs.
  • NT not tested.
  • Assay sensitivity was measured using dilutions of preparations of MLV (murine leukaemia virus) , SMRV (squirrel monkey retrovirus) and PoERV (porcine endogenous retrovirus) containing a known number of retroviral particles.
  • MLV murine leukaemia virus
  • SMRV squirrel monkey retrovirus
  • PoERV protein endogenous retrovirus
  • Purified RT enzyme was detected at 10 1 molecules for AMV and MLV using F-PERT. These levels of detection of purified enzymes are consistent with the data derived from virion particles, assuming a range of approximately 40-100 molecules per particle (Panet et al . , 1975; Krakower et al . , 1997).
  • the Gammaretrovirus PoERV has received considerable attention owing to the possible intimate contact with the human host during xenotransplantation (Patience et al . , 1997; Patience et al . , 1998; Takeuchi et al . , 1998).
  • the present F-PERT assay was optimised to include the detection 15 of the PoERV reverse transcriptase enzyme.
  • the present F-PERT assay can allow the discrimination of RT generated by infectious extracellular particles, and any RT activity that is associated with the endogenous transcription of the provirus pol gene inside the infected cell nucleus without production of viral particles.
  • the present F-PERT assay was able to detect endogenous retroviral activity from BHK-21, CHO, C127I, Mus dunni and Sf-9 cell supernatants.
  • Endogenous retroviral particles with reverse transcriptase activity have been detected in numerous species including invertebrates (Gabriel and Boeke, 1993; Hajek and Friesen, 1998), Chinese and Syrian hamsters (Anderson et al. , 1991; Dinowitz et al., 1992; Adamson, 1998; DeHaven et al., 1998), feline (Baumann et al . , 1998), murine (Wolgamot et al . , 1998) and Homo sapiens (Berkhout et al . , 1999).
  • Mammalian cell lines were cultured at 37°C in foetal calf serum supplemented Dulbecco's modified eagles medium (DMEM) or RPMI 1640 medium (Life Technologies Ltd. , UK) . Insect cells were cultured in SF900-II medium (Life Technologies Ltd. , UK) . Supernatants were harvested from the growing cultures when healthy, normally 2-5 days at 37°C post subculture and stored at or below a temperature of -70°C. 16 For induction of retrovirus from cell lines, duplicate cell cultures were initiated in tissue culture medium and after 24 hours 5-iodo-2-deoxyuridine (IDU; Sigma-Aldrich Co.
  • IDU 5-iodo-2-deoxyuridine
  • retroviruses used in this study are indicated in Figure 2.
  • An aliquot of each retrovirus was inoculated onto the appropriate detector cell lines, in the presence of lO ⁇ g/ml polybrene (Sigma-Aldrich Co. Ltd) , incubated at 37°C for approximately 90 minutes and fed with appropriate culture medium.
  • the inoculated cultures were maintained for 2-5 passages and supernatants harvested 2-5 days post subculture. Supernatants were centrifuged to remove cellular debris, filtered through a 0.45 ⁇ m filter unit, and stored in single use aliquots at or below -70°C.
  • Viral particles were pelleted by ultracentrifugation at 100,000 g for 60 min, followed by resuspension in DMEM (Life Technologies Ltd.). A selected volume of sample was mixed with an equal volume of latex spheres of known titre (Agar 17 Scientific) and diluted if necessary. The sample was then applied to a pioloform-coated EM mesh grid and allowed to air dry. The grid was fixed with 2.5% (v/v) glutaraldehyde, stained with 5% uranyl acetate and allowed to air dry completely. Samples were visualised using a Philips EM-400 transmission electron microscope.
  • Retrovirus particles were recognised by their size (80- 120nm) and structural features (concentric circles containing a dark inner core) . Latex spheres and retrovirus-like particles (rVLPs) were counted in parallel until 1000 latex beads were enumerated and the total particle count of the original volume calculated and retrovirus concentrations were recorded.
  • rVLPs retrovirus-like particles
  • Each pellet was resuspended in disruption buffer (40 mM Tris-HCl pH 8.1, 50 mM KCl, 20 mM dithiothreitol, 0.2% (v/v) NP-40) (Sigma-Aldrich Co. Ltd) and appropriate dilutions prepared. Typically 5-25 ⁇ l volumes of samples 18 were used immediately in RT reactions.
  • disruption buffer 40 mM Tris-HCl pH 8.1, 50 mM KCl, 20 mM dithiothreitol, 0.2% (v/v) NP-40
  • Cell lysates were prepared by suspending washed cell pellets in 200 ⁇ l of disruption buffer containing a CompleteTM EDTA-free protease inhibitor cocktail (Boehringer Mannheim, Cat. No. 1-836-170) , and incubated on ice for 20 min. Total cellular protein concentration was measured using a protein assay kit (Sigma Diagnostics Cat. No. P5656) . RT reactions were carried out with 25 ⁇ g, 10 ⁇ g, 1 ⁇ g and 100 ng of total cell lysate protein.
  • Synthetic oligonucleotide primers and probes used in the present F-PERT assay for Brome mosaic virus RNA and MS2 RNA were obtained from PE Biosystems Ltd. UK: Brome mosaic virus:
  • the fluorescent 5' -FAM (6-carboxy fluoresceine) and 3 1 - TAMRA (6-carboxy-N, N, N' ,N' -tetramethyl rhodamine) quencher labeled probe was as follows:
  • calf-thymus DNA polymerase ⁇ (Cambio, UK) , human DNA polymerase ⁇ (Chimerx, Millwaukee, USA) , calf liver polymerase y (Rai ond Lugert, Paul-Ehrlich-Institut, Langen, Germany) , DNA polymerase ⁇ and PCNA (Dmitry Mozzherin, State University of New York at Stony Brooke, USA) , native Taq DNA polymerase and terminal deoxynucleotide transferase (Life Technologies Ltd., UK).
  • Purified reverse transcriptase enzymes used in the study were Avian myeloblastosis virus (AMV: molecular weight 160kD, specific activity 98210 units/mg, Boehringer Mannheim cat. #. 1495-062) and recombinant Moloney-Murine leukaemia virus (M-MLV: molecular weight 71kD, specific activity 4 x IO 4 units/mg, Boehringer Mannheim cat. #. 1062- 20 603 ) .
  • AMV molecular weight 160kD, specific activity 98210 units/mg, Boehringer Mannheim cat. #. 1495-062
  • M-MLV molecular weight 71kD, specific activity 4 x IO 4 units/mg, Boehringer Mannheim cat. #. 1062- 20 603
  • Biosystems uses the 5' -nuclease activity of AmpliTaq GoldTM DNA polymerase to cleave a probe consisting of an oligonucleotide with a reporter dye at the 5' -end and a quencher dye at the 3' -end.
  • the quencher is only cleaved from the probe when the probe is hybridised with the target DNA and the increase in fluorescence of the probe can be measured.
  • the system detects and calculates a reporter dye value (R n ) for each sample during each cycle of amplification.
  • the value of R n or the normalised reporter signal, represents the fluorescence of the reporter dye divided by the passive reference dye.
  • R is the 5' -nuclease activity of AmpliTaq GoldTM DNA polymerase to cleave a probe consisting of an oligonucleotide with a reporter dye at the 5' -end and a quencher dye at the 3' -end.
  • ⁇ R. represents the normalised reporter signal R lid minus the baseline signal established in the first few cycles of PCR.
  • ⁇ R n increases during PCR as amplicon copy number increases until the reaction reaches a plateau.
  • the C ⁇ , or threshold cycle value is calculated in real time and represents the PCR cycle at which an increase in reporter fluorescence above the baseline signal can first be detected. The greater the amount of PCR template the lower 21 the threshold cycle (PE Biosystems TaqMan® Gold RT-PCR kit, 1997) .
  • Reagents used in PCR amplification were either the TaqMan® Universal PCR Master-mix (PE Biosystems #P/N 4304437) or the TaqMan® Gold RT-PCR kit (PE Biosystems # P/N808-0233) .
  • Amplification was quantified in real time by measuring the accumulation of fluorescent signal obtained from the 5' -nuclease activity of AmpliTaq GoldTM and the release of the reporter dye from the TaqMan® probe during the standard amplification cycles.
  • RT activity is expressed using 95% confidence limits of threshold cycle (C ⁇ ) values, where C ⁇ is the cycle at which a statistically significant increase in fluorescence is first detected.
  • RT activity was measured from cell line lysates and culture supernatants and purified reverse transcriptase enzymes using the exogenous RNA template from the Brome mosaic Virus (BMV) (Promega, UK) or bacteriophage MS2 (Boehringer Mannheim) .
  • BMV Brome mosaic Virus
  • MS2 Bacillus Virus
  • reverse transcriptase reactions the TaqMan® Gold RT-PCR kit (PE Biosystems # P/N808-0233) was prepared at 2X concentration.
  • test samples prepared in disruption buffer were added to the 2X RT reaction mix resulting in a final concentration of 50 mM KCl, 10 mM Tris-HCl pH8.3, 5.5 mM 22 MgCl 2 , 500 ⁇ M dATP, dCTP, dGTP and dTTP, 0.2 ⁇ M reverse primer, 0.1% (v/v) NP-40 (Sigma-Aldrich Co. Ltd), lOmM dithiothreitol (Sigma-Aldrich Co. Ltd), 0.4U/ ⁇ l placental RNAguard (Amersham Pharmacia Biotech; #27-0815-01) .
  • RT reactions contained BMV RNA (Promega) to aCT (Sigma-Aldrich Co. Ltd) ratios of 1:26, 1:52, 1:104, 1:208, 1:416 and 1:832 when appropriate (157 ng/ ⁇ l final concentration of aCT DNA in the RT reaction) .
  • Separate air spaces were used for the reagent preparation, test sample and spiked test sample processing, and amplification of cDNA products (Ou et al . , 1991).
  • a minimum of three replicates were used for each reaction in a 96 well plate format.
  • Sentinel controls for airborne contamination consisted of open wells containing the reagents described above and an equal volume of disruption buffer (Saksena et al . , 1991).
  • the mixes were incubated in an ABI 7700 sequence detection system at 48°C for 30 min for the RT step. Then the PCR reaction mix was added to each tube and the standard amplification was done: 10 min at 95°C to denature nucleases followed by forty amplification cycles of 15 sec at 95°C and 1 min at 60°C (PE Biosystems 1997) .
  • RT activity was quantified in real time by measuring the accumulation of fluorescent signal obtained from the 5 ' -nuclease activity of AmpliTaq GoldTM and the release of the reporter dye from the TaqMan® probe during amplification. RT activity is expressed using 95% confidence limits of threshold cycle (C ⁇ ) values, where C ⁇ is the cycle at which a statistically significant increase 23 in fluorescence is first detected.
  • the conventional RT assay measured the activities of reverse transcriptase with poly(rA) -oligo(dT) and the activities of contaminating DNA polymerase with poly(dA)- oligo(dT) (Temin and Baltimore, 1972; Kacian and Speilgelmen, 1974; Kornberg, 1980).
  • Retrovirus samples prepared as described above were added to equal volumes of reaction mixes containing 1 mM MnCl 2 (Sigma-Aldrich Co. Ltd), 0.025 A 26o units of poly(dA) or poly(rA) (Amersham Pharmacia Biotech), 80 mM Tris-HCl pH 8.0 (Sigma- Aldrich Co. Ltd), 100 mM KCl (Sigma- Aldrich Co.
  • Assay sensitivity was measured using dilutions of retroviral preparations containing a known number of retroviral particles.
  • dilutions of purified RT enzyme from AMV and M-MLV were tested in the range of lO ⁇ lO 1 molecules per reaction.
  • F-PERT reactions were done on dilutions of identical stocks of MLV, PoERV and SMRV with retrovirus particle counts of 2.9 x IO 6 , 2.0 x IO 8 and 2.8 x IO 7 per ml respectively.
  • the mean coefficient of variation for the particle counting assay by transmission EM was 16.7%.
  • the present F-PERT assay was able to detect 25 IO 2 MLV and IO 1 SMRV.
  • Reverse transcriptase activity when assayed in conventional RT assays using incorporation of radiolabelled nucleotides is dependent on the presence of divalent cations, either Mn 2+ or Mg 2+ , in the reaction mix (Temin and Baltimore, 1972; Klement and Nicolson, 1977).
  • divalent cations either Mn 2+ or Mg 2+
  • F-PERT F-PERT
  • compared these to those obtained from conventional RT and PERT The results demonstrated that the RT activity is greatly reduced if the alternative cation is used during the conventional RT assay.
  • the F-PERT assays were done in the presence of 11 mM and 5 mM Mg 2+ respectively and detected different retroviral RT with increased sensitivity regardless of cation preference ( Figure 4) .
  • Several studies have compared detection of different viruses in conventional RT and PERT assays, and demonstrated the high sensitivity and specificity of PERT (Pyra et al . , 1994; Heneine et al . , 1995; Yamamoto et al . , 1996;).
  • Detection of PoERV by conventional RT and C-PERT assays was relatively insensitive. Therefore the optimal conditions for the detection of the Mn 2+ dependent retrovirus PoERV were examined by assessing the effect of varying Mn 2+ and Mg 2* concentrations in the RT reaction (Phan- Thanh et al , 1992).
  • the F-PERT assay was performed on 10 s rVLPS of PoERV and MLV in the presence of 0.15-2 mM Mn 2+ . 27
  • the PoERV RT was also tested at varying concentrations of Mg 2 * cation.
  • the RT activity of MLV was shown to be similar at all Mn 2+ and Mg 2+ divalent cation concentrations tested.
  • the extract was then analysed at this level along with an equivalent activity of retroviral RT from IO 3 SMRV rVLPs.
  • an equivalent activity of retroviral RT from IO 3 SMRV rVLPs By decreasing the amount of BMV RNA template in two-fold series, conditions were established where signals from the cellular polymerases were suppressed, leaving the detection of RT activity unaffected. Elimination of any positive signals produced by the cellular polymerases from the retrovirus negative cell line MRC-5 was achieved with 15 ng of BMV per reaction. Positive activity was not observed in the absence of aCT DNA at 15ng of BMV RNA per reaction and below, indicating that under certain testing conditions RT- like DNA polymerase activity may be dependent on a higher concentration of BMV template in the RT reaction.
  • F-PERT is between three and six log 10 more sensitive than conventional RT assays and can detect a wide range of retroviruses (Figure 4) , including representatives of the Deltaretrovirus family (formally Type-D retroviruses) (Squirrel monkey retrovirus; SMRV, Simian retrovirus / SRV- 1) and Gammaretrovirus family (formally Type-C retroviruses) (Murine leukaemia virus; MLV, Porcine endogenous retrovirus; PoERV) , Lentivirus (Simian immunodeficiency virus; SIV) , and Spumavirus (Simian foamy virus; SFV) .
  • RT activity was not detected in non-induced Vero, Raji, MRC-5, BT, Mus dunni and C127I cell supernatants.
  • Treatment of Mus dunni and C127I cells with IDU resulted in induction of RT activity, indicating the presence of retroviral particles.
  • RT activity was detected in non-induced Sf-9, K-Balb, NSO, SP2/0-Agl4 and BHK-21 cell supernatants.
  • Induction of the endogenous retrovirus from K-BalbC cells resulted in a significant increase in RT activity in cellular supernatant.
  • the F-PERT assay inhibits the RT-like activity produced by cellular DNA polymerases of Vero and Mus dunni cells, further supporting the suppression of false positive signals by aCT DNA.
  • RT activity is not detected in cellular lysates and supernatants from MRC-5, BT, VERO, or Raji cells, whereas RT activity was detected in C127I, Mus dunni , K-Balb, BHK-21, SP2/0-Agl4 and NSO cell supernatants. RT activity was also detected in the Spodoptera cell line Sf9. 31
  • CrFK feline kidney cells produce an RD114-like endogenous virus that can package murine leukemia virus-based vectors. J Virol. 72, 7685-7687.
  • Novel retroviral sequences are expressed in the epididymis and uterus of Syrian hamsters. J Gen. Virol.79, 2687-2694. 32
  • Lugert R. Konig H. , Kurth R. and T ⁇ njes R. R. 1996. Specific suppression of false positive signals in the product enhanced reverse transcriptase assay. Biotechniques, 20, 210-217. Martin, U. , Kiessig, V., Blusch J.H., Haverich A., von der Helm, K. , Herden T. and G. Steinhoff. 1998. Expression of pig endogenous retrovirus by primary porcine endothelial cells and infection of human cells. Lancet 352, 692-694.
  • Patience, C Takeuchi, Y. , and R.A. Weiss. Infection of human cells by an endogenous retrovirus of pigs. 1997. Nature Medicine 3, 282-286
  • Patience, C. Patton, G.S., Takeuchi, Y., Weiss, R.A., McClure, M.O., Rydberg, L. and M.E. Breimer. 1998. The Lancet 352, 699-701.

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Abstract

L'invention concerne un procédé permettant de détecter une activité de transcription inverse (TI), notamment un rétrovirus endogène porcin, dans un échantillon pour essai. L'invention concerne également un kit à utiliser dans la détection d'une activité de transcriptase inverse dans un échantillon.
EP00966304A 1999-10-09 2000-10-09 Essai de transcriptase inverse Withdrawn EP1220951A2 (fr)

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GBGB9923846.1A GB9923846D0 (en) 1999-10-09 1999-10-09 Reverse transcriptase assay
GB9923846 1999-10-09
PCT/GB2000/003880 WO2001027318A2 (fr) 1999-10-09 2000-10-09 Essai de transcriptase inverse

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CA2395353A1 (fr) * 1999-11-29 2001-05-31 Glaxo Group Limited Analyse continue de transfert d'energie de resonance a resolution temporelle de polymerases d'acide polynucleique
WO2005003388A2 (fr) * 2003-06-30 2005-01-13 Astrazeneca Ab Dosages par fluorescence de polymerase d'acide nucleique
KR102068136B1 (ko) 2010-04-16 2020-01-20 모멘텀 바이오사이언스, 리미티드 비-정제된 샘플에서 세포 생존력을 결정하는데에 유용한 효소 활성을 측정하기 위한 방법
US11193176B2 (en) * 2013-12-31 2021-12-07 Bio-Rad Laboratories, Inc. Method for detecting and quantifying latent retroviral RNA species
EP3245306A4 (fr) * 2015-01-16 2018-09-05 Takeda Vaccines, Inc. Détection d'une activité de transcriptase inverse contenue dans des particules
CN116694741A (zh) * 2018-08-08 2023-09-05 加利福尼亚大学董事会 用于跨非连续模板的有序和连续的互补DNA（cDNA）合成的组合物和方法
CN116064722A (zh) * 2022-11-14 2023-05-05 翌圣生物科技(上海)股份有限公司 一种检测逆转录酶活性的试剂盒及检测方法

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JPH06509477A (ja) * 1992-05-11 1994-10-27 シュープバッハ・イエルク 逆転写酵素の検出方法
US5817457A (en) * 1996-02-07 1998-10-06 Ma Bioservices, Inc. Methods and kits for detecting viral reverse transcriptase activity in a sample using an acidic pH or an elevated temperature
WO1997040167A1 (fr) * 1996-04-19 1997-10-30 Q-One Biotech Ltd. Retrovirus porcin

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