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WO2012104360A1 - Test rt-pcr pour la détection du virus de l'hépatite e aviaire - Google Patents

Test rt-pcr pour la détection du virus de l'hépatite e aviaire Download PDF

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Publication number
WO2012104360A1
WO2012104360A1 PCT/EP2012/051723 EP2012051723W WO2012104360A1 WO 2012104360 A1 WO2012104360 A1 WO 2012104360A1 EP 2012051723 W EP2012051723 W EP 2012051723W WO 2012104360 A1 WO2012104360 A1 WO 2012104360A1
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Prior art keywords
hev
primer
pcr
avian
oligonucleotide
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Ivana BILLIC
Salome TROXLER
Michael Hess
Anna MAREK
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Veterinaermedizinische Universitaet Wien
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Veterinaermedizinische Universitaet Wien
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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
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
    • 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
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q2549/00—Reactions characterised by the features used to influence the efficiency or specificity
    • C12Q2549/10—Reactions characterised by the features used to influence the efficiency or specificity the purpose being that of reducing false positive or false negative signals
    • C12Q2549/119—Reactions characterised by the features used to influence the efficiency or specificity the purpose being that of reducing false positive or false negative signals using nested primers

Definitions

  • the present invention relates to methods for the detection of the presence of avian hepatitis E virus in a sample and to diagnostic test kits for the detection of the presence of avian hepatitis E virus in a sample.
  • Avian Hepatitis E Virus is the causative agent of big liver and spleen disease (BLSD) and hepatitis -splenomegaly syndrome (HSS) which were reported in Australia, Northern America and Europe (24). It was found to be enzootic in chicken flocks in the USA (15). Also in apparently healthy chicken flocks a high prevalence of antibodies was detected and avian HEV was isolated (30). In Europe wide distribution of avian HEV was reported (2, 21, 22, 25, 28, 32).
  • Avian HEV has not been isolated from other species than chicken, so far. Experimentally it was shown that it is able to cross the species barrier and infect turkeys (31), but not rhesus monkeys (17). Together with human and swine HEV, avian HEV belongs to the genus Hepevirus within the new family
  • HEV Hepeviridae (7). Phylogenetic analysis revealed that avian HEV forms a separate genus, itself consisting of at least three different genotypes and a geographic distribution pattern was observed (2, 21). Recently, HEVs detected in Norway rats were genetically identified (18). Furthermore, HEVs have been reported in several other animal species (23).
  • ELISA antibody tests
  • RT-PCR reverse transcription-polymerase chain reaction
  • RT-PCRs are described for avian HEV (15, 27, 30, 31) but real-time RT-PCR (SYBR-Green or TaqMan) methods exist only for human and swine HEV (1, 8, 10, 19, 20, 26). Not all of them are quantitative and only one (1) uses in vitro transcribed RNA as a standard for quantification.
  • SYBR-Green based RT-PCR methods have been described by Mackay, I.M. et al. (5).
  • TaqMan based RT-PCR methods are a development by Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA).
  • avian HEV The genomic organization of avian HEV is similar to human and swine HEV, both consisting of three open reading frames (ORFs) and two short non coding regions (NCRs).
  • ORFs open reading frames
  • NCRs short non coding regions
  • the length of avian HEV genome (6.6 kb) is about 600 bp shorter than human and swine HEV genome and they share only approximately 50 to 60 % sequence identity (2, 15, 17, 27). Therefore the molecular methods for detection of mammalian HEV are not suited for avian HEV.
  • RNA viruses that have highly variable genomes
  • the choice of appropriate primer and probe binding sites is crucial.
  • RT-PCR methods for detection of avian HEV used so far are of unknown specificity for samples of different geographic origin (23).
  • Many RT-PCRs for detection of avian HEV RNA described in literature use primers especially designed to anneal to the sequence of the isolate that was used in the same study (3, 4, 11, 12, 16, 17, 31).
  • a two-step RT-PCR was developed, that could detect RNA in liver samples from BLSD affected flocks (27).
  • RT-PCR methods for detection of avian HEV RNA are based on degenerate primers, binding within ORFl (15) or on a nested approach applying degenerate primers binding within ORFl and ORF2, respectively (30). These primers have e.g. been developed for RT-PCR detection of isolates from Northern America. Additionally, primers have been developed for RT- PCR detection of European and Australian avian HEV (2, 21).
  • a disadvantage of the primers used is, that they have a high specificity for avian HEV strains of the same geographical origin, but therefore they may possibly not react with avian HEV strains of a different geographical origin. As a consequence, the presence of some avian HEV strains in chicken flocks as well as in biological material may remain unnoticed.
  • Detection of HEV in a sample can be done i.a. by performing the following method steps: a) performing a reverse transcriptase polymerase chain reaction (RT-PCR) of said sample using a primer set comprising a primer that comprises a stretch of at least 16 consecutive nucleotides from an oligonucleotide BLSV-HEVf having a sequence as depicted in SEQ ID NO.: 5 and a primer that comprises a stretch of at least 16 consecutive nucleotides from an oligonucleotide BLSV-HEVr having a sequence as depicted in SEQ ID NO.: 6 and
  • RT-PCR reverse transcriptase polymerase chain reaction
  • step (a) examining the RT-PCR amplification result of step (a)
  • the term "primer” is used to describe an oligonucleotide that is capable to recognise, and to bind to, a complementary polynucleotide and to act as an initiation point for nucleic acid synthesis or replication along a complementary strand.
  • probe refers to an oligonucleotide that is complementary to a polynucleotide of interest, and capable of forming, under hybridisation conditions, a duplex structure with that polynucleotide. A probe doesn't need to be fully complementary to the polynucleotide of interest, as long as the hybridisation conditions are such that annealing can take place in spite of the incomplete complementarities.
  • probe basically refers to a primer that additionally carries a specific marker.
  • a marker can i.a. be a fluorophore, such as the fluorophores used in e.g. the TaqMan probes (vide infra).
  • oligonucleotide is used herein to describe a short polymer of nucleic acids. Such short polymer would usually have a length of between 10 and 100 nucleic acids.
  • hybridisation conditions relates to conditions that allow the primer or probe to anneal to the polynucleotide of interest. These conditions depend on the temperature and the ionic strength of the solution in which hybridisation is to take place. Hybridization reactions and conditions are well-known in the art and are i.a. described in the standard laboratory manual by Maniatis/Sambrook (34).
  • PCR-techniques are equally well-known in the art and they are extensively described in standard laboratory manuals such as; Real-Time PCR: Current Technology and Applications (35), in PCR primers, a laboratory manual (36), and in Maniatis/Sambrook (34).
  • the primers and the probe according to the invention can inter alia be used for the detection of avian HEV in an animal or a sample of that animal.
  • sample is used herein to refer to any biological material that is suspected of containing avian HEV.
  • the biological material can i.a. be tissue such as chicken liver, spleen, bone or muscle tissue, but it can also be a body fluid such as e.g. blood, urine, faecal material, amniotic fluid.
  • the material can also be a cloacal, oral or nasal cavity swab or e.g. disrupted cells.
  • a reverse transcriptase polymerase chain reaction comprises two reaction steps. In a first step, one of the primers of the primer set is allowed to bind to HEV-RNA, and this complex forms the starting point for the synthesis of a cDNA strand of the RNA strand by the enzyme reverse transcriptase (an RNA-dependent DNA polymerase) in the presence of the four DNA building blocks A, T, G and C.
  • reverse transcriptase an RNA-dependent DNA polymerase
  • RNA-DNA hybrid is heated in order to denature the hybrid, followed by cooling in order to allow the other primer of the primer set to bind to the cDNA strand.
  • This other primer then functions as the starting point for the synthesis of the second DNA strain by a DNA polymerase, again in the presence of the DNA building blocks.
  • the oligonucleotide BLSF-HEVf has the nucleotide sequence AAT GTG CTG CGG GGT GTC AA.
  • examples of a stretch of at least 16 consecutive nucleotides from BLSV-HEVf therefore are AAT GTG CTG CGG GGT G, T GTG CTG CGG GGT GTC and AAT GTG CTG CGG GGT GTC A.
  • a primer comprising a stretch of at least 16 consecutive nucleotides from an oligonucleotide having a sequence as depicted in SEQ ID NO.: means that the primer should at least have the length of a stretch of at least 16 consecutive nucleotides from the oligonucleotide as depicted in that SEQ ID NO.
  • the primer BLSV-HEVf has the sequence aatgtgctgc ggggtgtcaa as depicted in SEQ ID NO.: 5.
  • a "primer comprising a stretch of at least 16 consecutive nucleotides from an oligonucleotide having a sequence as depicted in SEQ ID NO.:” should at least consist of a stretch of at least 16 consecutive nucleotides from the nucleotides aatgtgctgc ggggtgtcaa in that order. It could however be a longer primer that e.g. comprises the nucleotides aatgtgctgc ggggtgtcaa, and has one or more additional nucleotides at the 5 '-end and/or the 3 '-end.
  • the oligonucleotide of the probe as depicted in SEQ ID NO.: 8 has a length of 22 nucleic acids, but again, the oligonucleotide of a probe according to the invention should have a minimal length of at least 16 consecutive nucleotides from an oligonucleotide having a sequence as depicted in SEQ ID NO.: 8.
  • a primer or probe
  • such nucleotides may or may not be complementary to the 3'- and/or 5 '-flanking regions of the complementary strand to which the primer binds.
  • the temperatures of the various RT-PCR cycles should possibly be adapted to the increased length of the primer and to the fact that one or more of the additional nucleotides are complementary.
  • the skilled artisan would be able, on the basis of the sequences of the primers and the probe, to determine the optimal temperature conditions for the various steps of the PCR-cycle using e.g. the formulae given in PCR text books referred to herein (vide supra).
  • Primers comprising a stretch of 17, 18, 19 or even 20 consecutive nucleotides from an oligonucleotide having a sequence as depicted in SEQ ID NO.: 5 or 6 are preferred in this order of preference, since they anneal even more selectively to avian HEV-sequences.
  • Primers or probes comprising a stretch of 17, 18, 19, 20, 21 or even 22 consecutive nucleotides from an oligonucleotide having a sequence as depicted in SEQ ID NO.: 8 are preferred in this order of preference, since they also anneal even more selectively to avian HEV-sequences.
  • step b) of the method of the present invention there are now different ways to perform step b).
  • the PCR step results in a PCR product of which the length and amount can be examined e.g. by means of conventional agarose or polyacrylamide gel electrophoresis.
  • the primer pair would anneal and therefore, after step a), a PCR product of the expected length would be detected on gel. If there is no avian HEV RNA present in the sample, the primer pair, or at least one of the primers, would fail to anneal and therefore, no PCR product of the expected length would be detected.
  • SYBR Green is a dye that intercalates with double-stranded DNA. This intercalation causes SYBR Green to fluoresce. Therefore, if the RT-PCR reaction is done in the presence of SYBR Green, each new cDNA copy would pick up an amount of SYBR Green and cause it to fluoresce.
  • a qPCR machine can detect the fluorescence and software can calculate Ct values from the intensity of the fluorescence. This allows for a direct quantification of the amount of cDNA made. (The use of SYBR Green however does not allow for the presence of an internal control that indicates if the reaction steps proceeded as expected).
  • the primer set BLSV-HEVf/BLSV-HEVr according to the invention is capable of giving a PCR-product in all cases, i.e. with all field isolates tested, regardless their
  • probe Probe HEV-3 (vide infra) is capable of detecting the PCR- product in all cases, i.e. with all field isolates tested, in a real-time PCR.
  • the method above thus provides a way of selectively detecting the presence or absence in a sample, of avian HEV, regardless the geographic origin of the HEV.
  • a first embodiment of the invention relates to a method for the detection of the presence of avian hepatitis E virus (avian HEV) in a sample, characterised in that said method comprises the steps of a) performing a reverse transcriptase polymerase chain reaction (RT-PCR) of said sample using a primer set comprising a primer that comprises a stretch of at least 16 consecutive nucleotides from an oligonucleotide BLSV-HEVf having a sequence as depicted in SEQ ID NO.: 5 and a primer that comprises a stretch of at least 16 consecutive nucleotides from an oligonucleotide BLSV-HEVr having a sequence as depicted in SEQ ID NO.: 6 and
  • RT-PCR reverse transcriptase polymerase chain reaction
  • step (a) examining the RT-PCR amplification result of step (a)
  • one primer of the primer set is the oligonucleotide BLSV-HEVf having a sequence as depicted in SEQ ID NO.: 5 and the other primer of the primer set is the
  • oligonucleotide BLSV-HEVr having a sequence as depicted in SEQ ID NO.: 6.
  • a nested set PCR One frequently used method for the detection of a specific nucleic acid sequence in a sample is known as a nested set PCR.
  • a PCR reaction is comparable to the PCR reaction described above, but it uses the PCR product made by the first primer set as a template for a second PCR reaction.
  • one of the two primers of the second primer set can be a primer that comprises a stretch of at least 16 consecutive nucleotides from the oligonucleotide of Probe HEV-3 having a sequence as depicted in SEQ ID NO.: 8.
  • this oligonucleotide also specifically binds to avian HEV cDNA, regardless the geographical origin of the avian HEV. This is even more remarkable because this oligonucleotide is slightly degenerate. Primers and probes on the basis of such degenerate oligonucleotides are in general slightly less selective due to their slightly degenerated character. In this case however, the degenerate character of this specific probe allows for selective binding only to avian HEV cDNA while at the same time allowing to bind to avian HEVs that slightly differ in the region to which this probe binds. Therefore, this oligonucleotide in combination with the primer set according to the invention allows for a very specific detection of avian HEV regardless the geographical origin of that avian HEV.
  • this oligonucleotide is used as a classical primer; so without the marker that is attached to the primer when it is used as a probe.
  • the oligonucleotide of the probe Probe HEV-3 anneals to the cDNA from position 4767-4788, which is close to the 5 '-terminus of the cDNA. Therefore, basically the only tool further needed for such a nested set PCR would be a second primer that is located close to the 3 '-terminus of the cDNA, i.e. around position 4900.
  • the use of a combination of the probe according to the invention as one primer and that second primer as the other primer would provide the basis for the nested set PCR.
  • Such a second primer can easily be designed by selecting an oligonucleotide e.g. around position 4900 and having a length of about 22 nucleotides, so e.g. from position 4897-4918, preferably an oligonucleotide having an AT/CG ratio is roughly comparable with that of the probe according to the invention.
  • an oligonucleotide e.g. around position 4900 and having a length of about 22 nucleotides, so e.g. from position 4897-4918, preferably an oligonucleotide having an AT/CG ratio is roughly comparable with that of the probe according to the invention.
  • Dieffenbach & Dreksler PCR primers, a laboratory manual. ISBN 0-87969-447-5 (1995) provide ample guidance when designing such a second primer.
  • the nested set PCR would result in a PCR product of which the length and amount can be analysed e.g. by means of conventional agarose or polyacrylamide gel electrophoresis.
  • the second primer pair formed e.g. by the oligonucleotide having a sequence as depicted SEQ ID No.: 8 and the second primer, would not, or at least not both, anneal to the first PCR product and thus there would be no PCR product of the expected length be detected on gel.
  • the nested set method above provides a way of more selectively detecting the presence or absence in a sample, of avian HEV.
  • a preferred form of this embodiment relates to a method for the detection of the presence of avian hepatitis E virus (avian HEV) in a sample, characterised in that said method comprises the steps of a) performing a reverse transcriptase polymerase chain reaction (RT-PCR) of said sample using a primer set comprising a primer that comprises a stretch of at least 16 consecutive nucleotides from an oligonucleotide BLSV-HEVf having a sequence as depicted in SEQ ID NO.: 5 and a primer that comprises a stretch of at least 16 consecutive nucleotides from an oligonucleotide BLSV-HEVr having a sequence as depicted in SEQ ID NO.: 6, followed by the step of
  • PCR polymerase chain reaction
  • step b examining the PCR amplification result of step b
  • one primer of the primer set is the oligonucleotide BLSV- HEVf having a sequence as depicted in SEQ ID NO.: 5 and the other primer of the primer set is the oligonucleotide BLSV-HEVr having a sequence as depicted in SEQ ID NO.: 6.
  • one primer of the second primer set is an
  • oligonucleotide having the sequence as depicted in SEQ ID NO.: 8.
  • step b) of the nested set PCR is to eliminate false positive signals, i.e.; in the unlikely case that the two primers of the first primer set according to the invention would pick up a non- avian HEV sequence or any other sequence from an avian sample, such false positive signals should not give a reaction in step b).
  • a nested set PCR increases the sensitivity of the test.
  • This method basically relies on the RT-PCR methods described above, but it does not even need the nested set PCR step. Nevertheless, it has a level of selectivity that compares to the nested set methods described herein. It relies only on the primer set according to the invention and a probe that comprises a stretch of at least 16 consecutive nucleotides from an oligonucleotide Probe HEV-3 having a sequence as depicted in SEQ ID NO.: 8, according to the invention. In this method, the probe is an oligonucleotide with a fluorophore and a quencher molecule attached to it.
  • probes are commercially available as TaqMan probes, Scorpions probes and Molecular Beacons probes (vide infra).
  • the detection can be done e.g. by using the probe according to the invention.
  • This probe binds, as said above, in a selective manner with an internal sequence of the cDNA made using the primer set according to the invention.
  • the probe anneals in step b) to the cDNA from position 4767- 4788, which is close to the 5'-terminus of the cDNA.
  • the probe according to the invention makes the detection of avian HEV more reliable than a mere RT-PCR reaction. In the unlikely case that the two selective primers would pick up a false positive signal, this would be noticed in step b/c, because the probe would not anneal to it.
  • TaqMan based real-time RT-PCR methods are a development by Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA.
  • TaqMan based real-time RT-PCR methods are described i.a. in literature references 8, 10 and 33.
  • TaqMan probes such as the Probe HEV-3 consist of a fluorophore covalently attached to the 5 '-end of the oligonucleotide probe and a quencher at the 3 '-end.
  • fluorophores e.g. 6- carboxyfluorescein, acronym: FAM, or tetrachlorofluorescin, acronym: TET
  • quenchers e.g.
  • TAMRA tetramethylrhodamine
  • MGB dihydrocyclopyrroloindole tripeptide minor groove binder
  • TaqMan probes are designed such that they anneal within a DNA region amplified by a specific set of primers. As Taq polymerase extends the primer and synthesizes the nascent strand, the 5' to 3' exonuclease activity of the polymerase degrades the probe that has annealed to the template. Degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in the realtime PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR. Taqman probes are preferred probes for use in the methods and diagnostic tools according to the invention.
  • the Taqman probe Probe HEV-3 described here is an oligonucleotide having a DNA sequence as depicted in SEQ ID NO.: 8, however with a fluorophore and a quencher attached to it. But as explained (vide supra), the probe can also be a shorter or longer oligonucleotide comprising a stretch of at least 16 consecutive nucleotides from an oligonucleotide Probe HEV-3 having a sequence as depicted in SEQ ID NO.: 8.
  • another preferred form of this embodiment relates to a method for the detection of the presence of avian hepatitis E virus (avian HEV) in a sample, characterised in that said method comprises the steps of a) performing a reverse transcriptase polymerase chain reaction (RT-PCR) of said sample using a primer set comprising a primer that comprises a stretch of at least 16 consecutive nucleotides from an oligonucleotide BLSV-HEVf having a sequence as depicted in SEQ ID NO.: 5 and a primer that comprises a stretch of at least 16 consecutive nucleotides from an oligonucleotide BLSV-HEVr having a sequence as depicted in SEQ ID NO.: 6 and
  • RT-PCR reverse transcriptase polymerase chain reaction
  • step (a) examining the RT-PCR amplification result of step (a) using a probe comprising a stretch of at least 16 consecutive nucleotides from an oligonucleotide Probe HEV-3 having a sequence as depicted in SEQ ID NO.: 8.
  • one primer of the primer set is the oligonucleotide BLSV- HEVf having a sequence as depicted in SEQ ID NO.: 5 and the other primer of the primer set is the oligonucleotide BLSV-HEVr having a sequence as depicted in SEQ ID NO.: 6.
  • the probe is Probe HEV-3.
  • This method according to the invention can be further improved by adding a so-called internal control RNA.
  • the internal control is a parallel experiment, in which an amount of a control RNA is added to the test sample, as well as primers and a probe that are specific for that control RNA (IC-RNA).
  • IC-RNA control RNA
  • the control RNA and the primers and probe are tested separately in a parallel RT-PCR test.
  • primers and probe should be non-HEV related; if they would be HEV-related they might interfere with the HEV-specific part of the method.
  • the IC-RNA test is performed in the same test tube as the avian HEV-detection test.
  • fluorescence of the IC-RNA specific fluorophore is detected, the test as such is reliable, and if in addition fluorescence of the HEV specific fluorophore is detected, that proves the presence of HEV-material.
  • an internal control is important to exclude false negative results due to e.g. incomplete RNA isolation, incomplete reverse transcriptase reaction or inhibition of PCR.
  • RNA can be used as the starting material for the internal control.
  • housekeeping genes or different genes of the host or from different pathogens can be used as internal control. Their unknown and changing concentration, instability and bio safety concerns make them however more difficult to handle and integrate in the PCR assay than in vitro transcribed RNA (14).
  • a preferred internal control system is the universal heterologous internal control system designed by Hoffmann et al. (14). It is based on RNA and could easily be adapted and integrated in the assay to check for successful RNA extraction and RT-PCR. As a longer product less inhibits amplification of target template (14), the primers for amplification of a 487 bp product were chosen ('AcGFP-13- F/AcGFP-10-R'). IC-RNA was added prior to RNA isolation, which resulted in a reliable amplification of internal control but did not inhibit amplification of avian HEV.
  • an even more preferred form of this embodiment relates to a method according to the invention, characterised in that the step of performing the real-time RT-PCR reaction of said method additionally uses at least one additional non-HEV related primer set and at least one additional non-HEV related probe. If quantification of the PCR reaction is required, separate parallel tests can be run in which known amounts of HEV-RNA and the primers and probe according to the present invention are present. This would allow for standard curves to be drawn that provide a relation between the amount of RNA in the parallel test and the number of cycles required to reach the fluorescence detection threshold. These standard curves can then subsequently be used to determine the unknown amount of HEV-RNA in the sample.
  • RNA for the internal control as well as the preparation of standard amounts of HEV-RNA for making standard curves in parallel tests are given in the Example section under the heading "Generation of standard and internal control RNA”.
  • this RNA is preferably purified from the sample to a certain extent.
  • Purification in this respect means that material in the sample other than HEV-RNA is to a certain extent removed from the sample before the sample is submitted to a method according to the invention.
  • Such purification can e.g. comprise de-proteinisation, removal of cell debris, removal of DNA, RNA-extraction and the like.
  • An example of RNA purification from a sample is given in literature reference 6. The Examples below also describe an efficient method to prepare purified RNA.
  • a still even more preferred form of the present invention relates to a method according to the invention, characterised in that said method comprises an RNA purification step preceding step a).
  • Another embodiment of the present invention relates to a primer set comprising a primer that comprises a stretch of at least 16 consecutive nucleotides from an oligonucleotide BLSV-HEVf having a sequence as depicted in SEQ ID NO.: 5 and/or a primer that comprises a stretch of at least 16 consecutive nucleotides from an oligonucleotide BLSV-HEVr having a sequence as depicted in SEQ ID NO.: 6
  • a further embodiment of the present invention relates to a primer set comprising a primer that comprises a stretch of at least 16 consecutive nucleotides from an oligonucleotide BLSV-HEVf having a sequence as depicted in SEQ ID NO.: 5 and/or a primer that comprises a stretch of at least 16 consecutive nucleotides from
  • a still further embodiment of the present invention relates to a primer or a probe comprising a stretch of at least 16 consecutive nucleotides from an oligonucleotide Probe HEV-3 having a sequence as depicted in SEQ ID NO.: 8 for use in the detection of the presence of avian HEV in a sample.
  • avian HEV avian hepatitis E virus
  • kits for the detection of the presence of avian hepatitis E virus (avian HEV) in a sample.
  • avian HEV avian hepatitis E virus
  • a first form of this second embodiment relates to a diagnostic test kit for the detection of the presence of avian hepatitis E virus (avian HEV) in a sample, characterised in that said kit comprises at least a primer set comprising a primer comprising a stretch of at least 16 consecutive nucleotides from an oligonucleotide BLSV-HEVf having a sequence as depicted in SEQ ID NO.: 5 and a primer comprising a stretch of at least 16 consecutive nucleotides from an oligonucleotide BLSV-HEVr having a sequence as depicted in SEQ ID NO.: 6
  • Another form of this second embodiment relates to diagnostic test kit for the detection of the presence of avian hepatitis E virus (avian HEV) in a sample, characterised in that said kit comprises
  • At least a primer set comprising a primer comprising a stretch of at least 16 consecutive nucleotides from an oligonucleotide BLSV-HEVf having a sequence as depicted in SEQ ID NO.: 5 and a primer comprising a stretch of at least 16 consecutive nucleotides from an oligonucleotide BLSV-HEVr having a sequence as depicted in SEQ ID NO.: 6 and at least a primer or a probe comprising a stretch of at least 16 consecutive nucleotides from an oligonucleotide Probe HEV-3 having a sequence as depicted in SEQ ID NO.: 8.
  • These various primers and probes may be present in the kit in separate vials.
  • a diagnostic test kit according to the invention may additionally comprise a reverse transcriptase and/or a thermo stable DNA polymerase. These enzymes are necessary to perform a real-time RT-PCR and for ease of use they might thus already be incorporated in the diagnostic test kit.
  • a second set of primers and probe in this case non-HEV primers and a non-HEV probe as discussed above, as well as the IC-RNA may be included in the diagnostic test kit. It goes without saying that the four DNA building blocks and the necessary buffers may additionally be included in the diagnostic test kit as well.
  • the diagnostic test kit additionally comprises known amounts of HEV-RNA that allow for quantification to be made.
  • RNA isolation was done accordingly to the 'Solution D' method (6), except that 96 % ethanol was used instead of 2-propanol for both precipitation steps (-80°C for 1 h, -20°C over night). If RNA isolation was performed with the use of internal control RNA, samples were first mixed with Solution D (lysis buffer) and then the internal control RNA was added. Further steps followed the usual protocol of the 'Solution D' method. Pellets were resuspended in 40 ⁇ ultra purified water and RNA was stored at -80°C.
  • Primer pairs 'Helicase F/Helicase R' (15) and 'Forwl C-BLSV/Revl C-BLSV (2) were used for conventional RT-PCR.
  • Primer pair 'BLSV- HEVf/BLSV-HEVr' and probe 'Probe ORF3-HEV were designed in this study and used for real-time RT-PCR.
  • In order to design these primers and probe four almost complete genome sequences of avian HEV (GenBank accession numbers AM943646, AM943647, AY535004, EF206691) were aligned using software Accelrys Gene, version 2.5 (Accelrys, San Diego, CA, USA).
  • primers and probes were tested in a BLAST search. All primers and probes were synthesized by Eurofins MWG Operon (Ebersberg, Germany).
  • PCR products were run on a 2 % agarose gel at 100 volts for 50 minutes and visualized on a UV illuminator.
  • a 176 bp RT-PCR product (primer pair 'BLSV-HEVf/BLSV-HEVr', Table 2), was cloned into pCR4-TOPO vector using a TOPO TA Cloning ® Kit for Sequencing (Invitrogen, Carlsbad, CA, USA).
  • RNA a 712 bp PCR product (primer pair 'AcGFP-15- F/AcGFP-10-R', Table 2) of the GFP gene using standard vector AcGFPl-Cl (Clontech Laboratories, Inc., Mountain View, CA, USA) was amplified and cloned. Positive clones were sequenced at Eurofins MWG Operon and clones of correct sequence and orientation were chosen for further processing.
  • the vectors were linearized using Pstl restriction enzyme (Invitrogen, Carlsbad, CA, USA) and run on a 1 % agarose gel for confirmation and purification. Bands of correct size were excised and cleaned using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) according to manufacturer's protocol. The purified plasmid DNA was then in vitro transcribed using the MAXIscript ® T7 kit (Ambion, Inc., Austin, TX, USA) according to manufacturer's protocol, except for adding 1 ⁇ RNaseOUT (Invitrogen, Carlsbad, CA, USA) to the transcription reaction, which was performed for 1 hour at 37°C.
  • RNA was treated with TURBO DNA-/reeTM Kit (Ambion, Inc., Austin, TX, USA) to completely remove residual DNA and precipitated with ammonium acetate and ethanol.
  • concentration of RNA was measured at least four times by a spectrophotometer (SmartSpec Plus, Bio-Rad Laboratories, Inc., Hercules, CA, USA) and copies per ⁇ were calculated using the mean values and the formula:
  • Real-time RT-PCR was performed in 25 ⁇ on Rotor-Gene Q (Qiagen, Hilden, Germany) using TaqMan detection method with QuantiTect ® Virus +ROX Vial Kit (Qiagen, Hilden, Germany).
  • primers and probe for avian HEV primers and probe for amplification and detection of IC-RNA were included in the mastermix.
  • the final mix was named 'HEV- 3/IC and consisted of primers 'BLSV-HEVf, 'BLSV-HEVr', 'AcGFP-13-F' and 'AcGFP-10-R', each in a final concentration of 0.4 ⁇ .
  • the probes 'Probe HEV-3 ' and 'Probe AcGFP' were added to the primer- probe mix 'HEV-3/IC in a final concentration of 0.2 ⁇ for each probe.
  • Duplex real-time RT-PCR was performed measuring simultaneously yellow and green fluorescence emitted by the two probes.
  • the conditions for RT-PCR were as follows: 50°C for 30 min, 95°C for 5 min, 50 times of three-step cycling with 95°C for 15 sec, 60°C for 75 sec and 72°C for 15 sec. Data analysis was done by Rotor-Gene Q software version 1.7 (Qiagen, Hilden, Germany) by setting the threshold automatically. In order to quantify samples of unknown concentration, their Ct values were compared with the standard curve and the copies of avian HEV RNA per reaction were calculated. Real-time RT-PCR products were run on a 2 % agarose gel to test if products of correct size were amplified. Reverse transcriptase control reactions were included in order to confirm that the in vitro transcribed RNAs were not contaminated with residual DNA.
  • HEV-RNA in vitro transcribed RNA from 'BLSV- HEVf/BLSV-HEVr' PCR product
  • the primer pair 'AcGFP- 13-F/AcGFP-10-R' that amplifies a 487 bp PCR product, was tested with dilution series of in vitro transcribed IC-RNA.
  • dilution series of IC-RNA had been made and the least amount detected together with HEV-RNA reliably was chosen for use in the future assay.
  • the ten-fold dilution series of in vitro transcribed HEV-RNA were spiked with 5.7 ⁇ 10 5 copies of in vitro transcribed IC-RNA. Since in the course of RNA isolation a certain amount of RNA is lost, 5.7 ⁇ 10 7 copies of IC- RNA were added to the samples to check the efficiency of RNA isolation.
  • RNA (07/861) was serially diluted ten-fold, TaqMan real-time RT-PCR (primer-probe mix 'HEV-3/IC') and conventional RT-PCR (primer pairs 'Helicase F/Helicase R' and 'Forwl C-BLSV/Revl C-BLSV') were performed and the results were compared.
  • RNA and DNA isolated from tissue culture were avian leukosis virus (RNA and DNA isolated from tissue culture), Marek's disease virus (DNA isolated from a positive field sample), avian reovirus (RNA isolated from vaccine) and fowl adenovirus (DNA isolated from tissue culture).
  • avian leukosis virus RNA and DNA isolated from tissue culture
  • Marek's disease virus DNA isolated from a positive field sample
  • avian reovirus RNA isolated from vaccine
  • fowl adenovirus DNA isolated from tissue culture
  • the duplex real-time RT-PCR assay showed a range over ten orders of magnitude (2.89 ⁇ 10 12 to 2.89 ⁇ 10 3 copies per reaction). It has to be mentioned that the smallest dilution (2.89 ⁇ 10 3 copies per reaction) was not detected reliably and mostly did not fit well into the standard curve. If this dilution was not taken into account, an efficiency of 1.04 and a regression square of 0.996 was achieved. The IC-RNA added to standard curve was detected reliably with an average Ct of 30.17 and a standard deviation of 0.79 (data not shown).
  • RNA of sample 07/861 A was diluted five times ten-fold and conventional and real-time RT-PCRs were performed. In conventional RT-PCR the dilutions up to 10 "3 were detected using both primer pairs,
  • the duplex real-time RT-PCR was performed with samples positive for swine HEV, wild boar HEV, rat HEV, avian leucosis virus, Marek's disease virus, avian reovirus, fowl adenovirus and a sample of RNA isolated from the liver of an SPF chicken. All samples were negative, i.e. no Ct was measured and no band was visible after gel electrophoresis. The internal controls and the avian HEV positive control reaction were positive. 2.5 Sample detection
  • the real-time RT-PCR assay based on the degenerate probe could detect all of the 16 samples tested (Table 4). In conventional RT-PCR nine out of 16 samples were detected with both primer pairs
  • probe-based detection systems such as TaqMan have an improved specificity (13).
  • the degenerate probe 'Probe HEV-3' could detect all samples tested in this study. Among these were samples that were negative in conventional RT-PCR. Therefore, it was concluded that the real-time RT-PCR based on 'BLSV-HEVf/BLSV-HEVr' primers and 'Probe HEV-3' is highly suited for detecting avian HEV samples of different geographical origins and genotypes.
  • the samples were obtained from poultry in different European countries and Australia and were mainly classified within genotypes 1 and 3 of avian HEV (21).
  • genotype 2 One sample belonging to genotype 2, according to analysis of helicase gene sequence, was also successfully tested with the real-time RT-PCR method. Therefore, the real-time RT-PCR according to the invention should also be suited for samples representing all different genotypes reported so far.
  • In vitro transcribed RNA was used as standard for quantification of avian HEV RNA, which is important because the reverse transcription step is limiting and amplification of RNA is not proportional to amplification of DNA, e.g. amplification of a plasmid used as standard.
  • the method according to the invention detected approximately 3.6 x 10 3 copies of in vitro transcribed HEV RNA.
  • the real-time RT- PCR for detection of avian HEV had a sensitivity similar to conventional RT-PCR using ten-fold dilutions of sample RNA. Not all samples were detected in conventional RT-PCR, although two primer pairs, binding within ORFl and ORF2, were used.
  • the real-time RT-PCR presented here has the advantage to detect a large range of samples from different genotypes in a single procedure step with only one primer pair, having the same sensitivity as conventional RT-PCR. Furthermore, it was possible to quantify the samples tested in this study by use of a standard curve consisting of in vitro transcribed RNA. All Ct values of the samples were in range of the standard curve and the amount of avian HEV present in each sample could be expressed as copies of avian HEV RNA per reaction. If quantification of samples is done, it must be considered that the values are theoretical and need normalization based on equal quantity and quality of starting material (9).
  • the TaqMan real-time RT-PCR method according to the invention is suitable for universal detection of avian HEVs.
  • the same was found to be true for the TaqMan duplex real-time RT-PCR method according to the invention.
  • the real-time RT-PCR method according to the invention is a specific and sensitive diagnostic tool for universal detection of avian HEV in animal samples.

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Abstract

La présente invention concerne des procédés pour la détection de la présence de virus de l'hépatite E aviaire dans un échantillon au moyen d'un ensemble d'amorces spécifiques et facultativement d'une sonde spécifique et des kits de test de diagnostic pour la détection de la présence du virus de l'hépatite E aviaire dans un échantillon qui comprennent un tel ensemble d'amorces spécifiques et facultativement une sonde spécifique.
PCT/EP2012/051723 2011-02-03 2012-02-02 Test rt-pcr pour la détection du virus de l'hépatite e aviaire Ceased WO2012104360A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2017137095A1 (fr) * 2016-02-12 2017-08-17 Curevac Ag Procédé d'analyse d'arn
EP3414340B1 (fr) * 2016-02-12 2023-08-09 CureVac SE Procédé d'analyse d'arn
US11920174B2 (en) 2016-03-03 2024-03-05 CureVac SE RNA analysis by total hydrolysis and quantification of released nucleosides
CN110878378A (zh) * 2019-12-02 2020-03-13 昆明理工大学 用于检测体液中戊型肝炎病毒的引物组合及其应用
WO2023234486A1 (fr) * 2022-06-03 2023-12-07 주식회사 세니젠 Kit pour pcr en temps réel pouvant détecter le virus de l'hépatite e

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