Classifications

• • 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

Definitions

• the invention relates to the fields of diagnostics and the copying and/or amplification of nucleic acid.
• the invention relates to means and methods for detecting virus using molecular biological tools.
• the present invention is exemplified by means of viruses, in particular human enteroviruses, adenoviruses and influenza.
• viruses in particular human enteroviruses, adenoviruses and influenza.
• the invention is by no means limited to enterovirus, adenovirus and influenza detection.
• Means and methods for isolating and reverse transcribing RNA are applicable using any type of RNA.
• EV Human enteroviruses
• poliovirus PV's; 1 to 3
• Coxsackieviruses A CAVs; 1 to 22 and 24
• Coxsackieviruses B CBVs; 1 to 6
• echoviruses 1 to 7, 9, 11 to 27, and 29 to 33
• enteroviruses 68 to 71 18
• echovirus 22 and 23 were genetically distinct from the members of the Enterovirus genus and were reclassified in a separate genus of Parechoviruses within the family of Picornaviridea (14, 21, 25). Infections with EV cause a wide range of clinical outcomes like asymptomatic infections, aseptic meningitis (meningeal inflammation in the absence of a bacterial pathogen), encephalitis, paralytic poliomyelitis, and myocarditis. Although the majority of EV infections do not cause significant disease, infection can cause serious illness, especially in infants and immune-compromised patients.
• EV infections are the most common cause of aseptic meningitis and account for 80 — 90% of all cases of CNS infection for which a possible causative agent is identified (26). In the neonate, aseptic meningitis induced complications and poor outcome of EV infections generally occur within the first 2 days of life (1, 3). Aseptic meningitis in immune -competent adults is characterized by sudden onset of fever but neurological abnormalities are rare and both short term and long term outcome are generally good. Encephalitis caused b3 ⁇ EV infections is a less common but a more severe disease than aseptic meningitis (19, 31, 32). Immune-compromised children and adults who are infected with EV may develop chronic meningitis and encephalitis, which maj ⁇ last years before becoming fatal (17).
• RT-PCR may become the diagnostic method of choice for EV infections of CNS, in many laboratories diagnosis of EV still relies on cell culture techniques. Stool specimens or rectal swabs, throat swabs, and CSF are used in virus culture.
• EV can be detected by RT-PCR in all kinds of clinical specimens like whole blood, plasma, serum, CSF, Stool specimens, throat swabs, vesicle fluids, pleuratic fluids, broncheo-alveolar-lavages, amniotic fluids, urine, and brain biopsies.
• CPE Cytopathic effect
• the invention therefore provides a method for reverse transcribing an RNA target molecule comprising incubating said RNA target molecule with a reverse transcriptase.
• the reverse transcriptase is a Moloney Murine Leukemia Virus Reverse Transcriptase [M-MLV RT], more preferably a reverse transcriptase known as Superscript II or III (RNase H- Reverse Transcriptase) described in Potter et al, Focus Vol 25.1; pp 19-24.
• Superscript II contains a series of point mutations in the Rnase H domain of M-MLV, or a functional part, derivative and/or analogue of said Superscript II or III, in a solution comprising between 2,5 and 7,5 mM of a suitable salt, preferably 5.0 niM, between 0.05 % and 0.2 % of a non-ionic detergent, preferably 0.1%, between 60 and 240 ⁇ M per nucleotide, preferably 120 ⁇ M and a suitable buffer that buffers said solution at a pH between about 8 or 10, said method further comprising incubating said solution at a temperature of between 25 and 50 °C.
• a functional part, derivative and/or analogue of superscript II or III comprises the same activity in kind not necessarily in amount as superscript II or III themselves under the above mentioned conditions.
• Suitable salts are salts of group I of the periodic system, preferably a sodium or potassium salt.
• the counter-ion in the salt is preferably chloride.
• the non-ionic detergent is non-denaturing and significantly improves both the yield and quality of the reverse transcriptase (RT) product.
• the non- ionic detergent is preferably non-idet p40, or triton X100, or a combination or an equivalent thereof.
• the non-ionic detergent is Triton X100 or an equivalent thereof.
• the non-ionic detergent is preferably present in amount of between about 0.05 and 0.2 % (vol/vol).
• the non-ionic detergent is present in an amount of about 0.1% (vol/vol).
• all four of different categories of nucleotides characterised by dATP, dCTP, dGTP and dTTP are added to the solution.
• One or more of the categories may be omitted, for instance when a specific typically short RT product is desired. However, at least one of the above mentioned categories is required in the solution.
• many different derivatives and analogues of the prototype nucleotides are available.
• Such derivatives and analogues when they can be incorporated into the nascent strand, can also be used in the present invention. It is also possible to add one or more so-called stopper nucleotides in the solution for, for instance, sequencing purposes.
• any nucleotide or derivative and/or analogue thereof can be used in the present invention.
• a particular category of nucleotide is typically present in an amount of between about 60 and 240 ⁇ M per nucleotide.
• a particular categoiy of nucleotide is present in an amount of between about 100 and 140 ⁇ M, more preferably in an amount of about 120 ⁇ M.
• the temperature range is typically a range wherein superscript is active.
• the temperature range is between about 25 and 50 °C. Preferably, between about 40 and 50°C. More preferably, the incubation temperature is about 42 °C for Superscript II or a functional part, derivative and/or analogue thereof and about 50 °C for Superscript III or a functional part, derivative and/or analogue thereof.
• the buffer can be any compound when it is suitable for use in enzymatic processes and capable of buffering a solution between about pH 8 and 10.
• buffers common in enzymatic processes in molecular biology are used.
• the buffer comprises TRIS or HEPES or a combination or equivalent thereof.
• the solution is buffered by said buffer at a pH of between about 8 and 10. Preferably between about pH 8 and 9.
• the solution is buffered by said buffer at an pH of about 8.3.
• Counter ions for adjusting the buffer to the desired pH are preferably sodium, potassium and chloride.
• a primer for the reverse transcriptase The RNA itself can act as a primer as, for instance, is the case with genomic retroviral RNAs. However, typically at least one primer is added to the solution.
• the primer can be any oligonucleotide or equivalent thereof that is capable of hybridising to the RNA of interest and capable of being elongated by the reverse transcriptase.
• primer oligonucleotide for RT priming comprises the same hybridisation and RT priming capability in kind not necessarily in amount.
• the primer oligonucleotide typically comprises between about 4 and 30 nucleotides or equivalent thereof.
• the primer typically contains between about 4 to 10 nucleotides or equivalent thereof.
• the primer typically contains between about 10 and 30 nucleotides or equivalents thereof, more preferably between about 15 and 25 nucleotides, preferably between about 18 and 22 nucleotides, more preferably about 20 nucleotides.
• the primer is a hexamer.
• the primer is a mixture of at least 5 and preferably at least 10 and more preferably at least 20 ohgonucleotides or equivalent thereof, wherein said oligonucleotides or equivalents thereof differ from each other by at least one nucleotide.
• the primer is a hexamer mixture comprising at least 20 of the mentioned different ohgonucleotides or equivalents thereof.
• the RNA may be derived from any sample.
• the invention provides a method of the invention further comprising preparing RNA target molecule from a sample comprising faeces, preferably human faeces.
• the RNA target molecule comprises sequences derived from an enterovirus, preferably a human enterovirus (EV) or an influenza virus, preferably influenza A.
• an enterovirus preferably a human enterovirus (EV) or an influenza virus, preferably influenza A.
• EV human enterovirus
• influenza virus preferably influenza A.
• a method of the invention further comprises amplifying produced RT product using one or more primers.
• EV-RNA was purified without a control (IC-RNA) that monitored both RNA extraction efficiency and RT-PCR efficiency.
• a method of the invention further comprises providing a sample containing the RNA target molecule with an internal control nucleic acid.
• this internal control nucleic acid comprises RNA. This may be done by adding the internal control to the sample or by adding the internal control already at an ear her stage, tor example to the faeces or the isolated RNA there from.
• said solution further comprises an internal control RNA molecule.
• In vitro transcribed RNA can be used as an IC-RNA but is prone to degradation by RNAses.
• the internal control nucleic acid comprises armoured RNA.
• the armoured RNA is prepared using the means and methods disclosed herein or disclosed in (24). Alternatively, or additionally, another method for at least in part avoiding degradation of RNA by RNAses is applied.
• the internal control RNA comprises a sequence 5'-CCC TGA ATG CGG CTA ATC CTA ACC ACG GAA CAG GCG GTC GCG AAC CAG TGA CTG GCT TGT CGTAAC GCG CAA GTC TGT GCT TGA GAC GTG CGT GGT AAC CGT CCG TGT TTC CTG TTA TTT TTA TCA TGG CTG CTTATG GTG ACAAT-3'.
• IC-RNA's should have about the same length, contain the same primer binding sites, and have about the same GC content for identical extraction and amplification efficiency but should contain a different probe binding site for the differential detection of IC-RNA.
• the internal control nucleic acid comprises about the same length, primer binding site(s) and GC content as the region in the EV RNA that is selected for reverse transcription and subsequent amplification.
• said internal control comprises at least one location where the nucleic acid encodes a sequence that differs from at least all human enteroviruses. Detection of this region, for instance, by means of a probe thus can distinguish between internal control and EV-RNA.
• the region that is selected for amplification comprises sequences that are conserved between human enteroviruses as this allows the use of one primer set for the detection of all (currently identified) enteroviruses.
• the selected region further comprises, a further location that is conserved between human enteroviruses, wherein the location is internal to the amplified region and does essentially not exhibit overlap with the location of the primer(s).
• one detection means such as a probe for the detection of essentially all (currently known) human enteroviruses.
• the invention provides a method wherein said internal control RNA molecule comprises a sequence 5'-CCC TGA ATG CGG CTA ATC CTA ACC ACG GAA CAG GCG GTC GCG AAC CAG TGA CTG GCT TGT CGT AAC GCG CAA GTC TGT GCT TGA GAC GTG CGT GGT AAC CGT CCG TGT TTC CTG TTA TTT TTA TCA TGG CTG CTT ATG GTG ACA AT-3'.
• the primer(s) for amphfication of this sequence allows amplification of essentially all human enteroviruses, whereas it further span a region that further comprises a further conserved region for the easj ⁇ detection of amplified material from all, currently known, human enteroviruses.
• This sequence, and in particular the choice of the primer binding sites has the additional advantage that, whereas assay in the art detect also human viruses related to the human enteroviruses, the assay of the invention is more selective as at least the closely related human rhinoviruses are not detected using an assay of the invention. This feature of course greatly improves the reliability of the assay of the invention.
• the invention further provides a method for testing a sample for the presence therein of a nucleic acid derived from an enterovirus, comprising providing a nucleic acid amplification solution with said sample, with a first primer comprising a nucleotide sequence ID#1: 5'- CCC TGA ATG CGG CTA AT-3' or a functional equivalent thereof and with a second primer comprising a nucleotide sequence (ID#2: 5'-ATT GTC ACC ATA AGC AGC C-3' or a functional equivalent thereof, and incubating said amplification solution to allow for amplification of said nucleic acid when present.
• a functional equivalent of a primer comprises the same 10 bases when measured from the 3'-end but contains a mismatch of preferably 2, and more preferably 1 nucleotide in the 10 bases when measured from the 5'-end. It has been found that such functional equivalents comprise essentially the same specificity and amplification efficiency as the original they are derived from.
• the 3' end of a functional equivalent of a primer of the invention is preferably identical to said primer, while the 5' end of said functional equivalent may differ from said primer such that the properties essentially remain the same in kind, not necessarily in amount. According to the invention, the 5' end of a primer of the invention can be varied while retaining the properties of said primer in kind, not necessarily in amount.
• a functional equivalent of a primer of the invention contains at most 30, preferably at most 25, more preferably at most 20, more preferably at most 15, most preferably at most 10 additional nucleotides at its 5' end as compared to said primer. Furthermore, at least one nucleotide of said functional equivalent niaj ⁇ be changed as compared to said primer, preferably in its 5' end region.
• Said sample tested with a method of the invention preferably comprises product of a method for reverse transcribing an RNA target molecule according to the invention.
• a method of the invention further comprises analysing the presence or absence of said nucleic acid by means of a probe.
• a probe comprising a sequence ID#3: EV-specific probe; 5'-GCG GAA CCG ACT ACT TTG GGT-3' or a functional equivalent thereof.
• a method for testing a sample of the invention further comprises analysing the presence or absence of an internal control nucleic acid by means of a probe comprising a sequence ID#4: IC-specific probe; 5'-CTT GAG ACG TGC GTG GTA ACC-3 or a functional equivalent thereof.
• Preferred primers, probes and internal control nucleic acid suitable for detecting enterovirus are depicted in Table 13.
• the invention furthermore provides primers, probes and internal control nucleic acids which are preferably used for testing a sample for the presence therein of a nucleic acid derived from viruses other than enteroviruses.
• Tables 6-12 depict primers, probes and internal control nucleic acids suitable for testing a sample for the presence therein of a nucleic acid derived from influenza A virus, influenza B virus, metapneumovirus, Respiratory Syncitial Virus, Rhino virus, Adenovirus and Parainfluenza virus.
• R means G or A
• S means G or C
• Y means T or C.
• a primer of the invention provides an improved result, even if a prior art primer is capable of annealing somewhere in the same kind of viral nucleic acid region as a primer of the present invention. This is for instance shown in example 4.
• a primer of the invention provides a more sensitive and/or more specific assay.
• the invention thus provides a method for testing a sample for the presence therein of a nucleic acid derived from a virus, comprising providing a nucleic acid amplification solution with at least part of said sample and with a first and a second primer, wherein said first primer comprises a nucleotide sequence of a forward primer depicted in any one of tables 6-13, or a functional equivalent thereof, and/or wherein said second primer comprises a nucleotide sequence of a reverse primer depicted in any one of tables 6-13, or a functional equivalent thereof, and incubating said amphfication solution to allow for amplification of said nucleic acid when present.
• a nucleic acid amphfication reaction is preferably provided with said sample and with at least one oligonucleotide comprising a sequence of a primer as depicted in Table 6, or at least one equivalent thereof.
• both primers of any one of tables 6-13, or at least one functional equivalent of at least one of said primers is used.
• a nucleic acid amplification solution is most preferably provided with said sample, with an oligonucleotide comprising a sequence of the forward primer depicted in Table 6, and with an oligonucleotide comprising a sequence of the reverse primer depicted in Table 6, or at least one functional equivalent of at least one of said primers.
• a nucleic acid amplification solution is preferably provided with said sample and with an oligonucleotide comprising a sequence of the forward primer depicted in Table 7, and with an oligonucleotide comprising a sequence of the reverse primer depicted in Table 7, or at least one functional equivalent of at least one of said primers.
• a nucleic acid amplification solution is preferably provided with said sample and with an oligonucleotide comprising a sequence of the forward primer depicted in Table 11, and with an ohgonucleotide comprising a sequence of the reverse primer depicted in Table 11, or at least one functional equivalent of at least one of said primers, et cetera.
• a method of the invention is provided wherein said virus comprises an enterovirus.
• said virus comprises an adenovirus or influenza virus, because these viruses are commonly found in individuals.
• One embodiment therefore provides a method of the invention, wherein said first primer comprises the sequence 5'-CAGGACGCCTCGGRGTAYCTSAG-3' or a functional equivalent thereof and said second primer comprises the sequence 5'-GGAGCCACVGTGGGRTT-3' or a functional equivalent thereof. These primers are particularly suitable for testing a sample for the presence of adenoviral nucleic acid.
• said first primer comprises the sequence 5'-GACAAGACCAATCCTGTCACYTCTG-3' or a functional equivalent thereof and said second primer comprises the sequence 5'-AAGCGTCTACGCTGCAGTCC-3' or a functional equivalent thereof. These primers are particularly suitable for testing a sample for the presence of influenza A nucleic acid.
• RNA is preferably converted into DNA. This is most preferably performed with a method of the present invention.
• Said sample therefore preferably comprises a product of a method for reverse transcribing an RNA target molecule according to the invention.
• a method of the invention further comprises analysing the presence or absence of viral nucleic acid by means of a probe.
• a probe comprising a nucleotide sequence of the virus -specific probe depicted in the same table as said forward primer and/or said reverse primer, or a functional equivalent thereof.
• a virus-specific probe as depicted in Table 6, or a functional equivalent thereof, is preferably used.
• a virus-specific probe of the same Table is preferably used.
• a method of the invention comprising analysing the presence or absence of viral nucleic acid with a probe comprising a nucleotide sequence of the virus-specific probe depicted in the same table as said forward primer and/or said reverse primer, or a functional equivalent of said probe, is therefore preferably provided.
• nucleic acid derived from an enterovirus is analysed with a probe comprising a sequence ID#3: EV-specific probe; 5'-GCG GAA CCG ACT ACT TTG GGT-3'or a functional equivalent thereof.
• nucleic acid derived from an adenovirus is analysed with a probe comprising a sequence 5'-CCGGGTCTGGTGCAGTTTGCCCGC-3'or a functional equivalent thereof.
• nucleic acid derived from an influenza A virus is analysed with a probe comprising a sequence
• an internal control nucleic acid is preferably used. If the presence of RNA derived from an RNA virus in a sample is investigated, said internal control nucleic acid preferably also comprises RNA. Likewise, if the presence of DNA derived from a DNA virus in a sample is investigated, said internal control nucleic acid preferably comprises DNA. This allows for accurate verification of assay sensitivity and for accurately avoiding false negative results. In one embodiment a method of the invention therefore further comprises providing said nucleic acid amplification solution with an internal control nucleic acid molecule.
• said internal control comprises at least one location where the nucleic acid comprises a sequence that differs from all viruses that are tested for.
• an internal control depicted in any one of tables 6-13 is used. If an ohgonucleotide comprising a primer sequence of one specific table (or a functional equivalent thereof) is used, it is preferred to use an ohgonucleotide comprising the internal control nucleic acid sequence of the same table (or a functional equivalent thereof).
• a combination of ohgonucleotides comprising at least one primer, probe and/or internal control nucleic acid sequence of any one of Tables 6-13, or at least a functional equivalent of said at least one primer, probe and/or internal control, enhances the sensitivity and/or specificity of a method of the invention.
• the invention therefore provides a method of the invention, wherein said internal control nucleic acid molecule comprises a nucleotide sequence of the internal control depicted in the same table as said primer, or a functional equivalent of said internal control.
• an internal control nucleic acid of the invention preferably contains the same primer binding sites, although this is not necessary.
• Said internal control nucleic acid furthermore preferably has essentially the same length and preferably has about the same GC content for essentially the same extraction and/or amplification efficiency but should contain a different probe binding site for differential detection of internal control nucleic acid. Detection of said internal control nucleic acid by a different IC-specific probe enables distinguishing between internal control and viral nucleic acid.
• an IC-specific probe comprising a sequence as depicted in Tables 6-13 is ised.
• the invention thus provides a method of the invention, further comprising analysing the presence or absence of said internal control nucleic acid b3 ⁇ means of a probe comprising a nucleotide sequence of the IC-specific probe depicted in the same table as said internal control, or a functional equivalent of said probe.
• said probe comprises the sequence 5'-CTT GAG ACG TGC GTG GTA ACC-3' , 5'-GATGTGTCCGCCGTGGTCCCCTGG-3', and/or
• 5'-TTCACTGGGCCCGACTCGCACTGAC-3' or a functional equivalent thereof, in order to test a sample for the presence of a nucleic acid derived from enterovirus, adenovirus and/or influenza A, respectively.
• For detection probes may be labelled by any means suitable. Many different methods are known in the art. Amplified product can be measured directly or indirectly through measurement of label associated with the probe. Amplified product may be measured at the end of the amplification step or in real-time during amplification. Such methods are known in the art. Real time measurement of amplified product is preferred because results are obtained faster with less effort, while the sensitivity is increased, as compared to end-point PCR, and the method is less prone to contamination.
• a probe preferably comprises a fluorophore. Other preferred labels include radio-active and/or digoxigenine labels. Label maj'- be associated directly to the probe, or the probe may be detected indirectly, through a labelled binding member specific for said probe.
• a sample is tested with a method of the invention for the presence therein of at least two different viruses. For instance, if an individual shows clinical signs of infection of the respiratory tract, it is preferred to test a sample of said individual for the presence of a variety of viruses capable of infecting the respiratory tract. In that case, a sample is preferably tested for the presence of nucleic acid of at least two viruses depicted in tables 6-13. Said two viruses preferably comprise Adenovirus and Influenza.
• a method of the invention is preferably performed using at least two ohgonucleotides comprising at least one primer, probe and/or internal control sequence depicted in a first table selected from the group consisting of tables 6- 13, or at least one functional equivalent thereof, and at least two ohgonucleotides comprising at least one primer, probe and/or internal control sequence depicted in a second table selected from the group consisting of tables 6-13, or at least one functional equivalent thereof.
• the invention provides a method of the invention, wherein a sample is tested for the presence of a first virus as depicted in any one of Tables 6-13 and for the presence of a second virus as depicted in any one of Tables 6-13, using at least two primers depicted in the same Tables as said first and said second virus.
• a sample is tested for the presence of nucleic acid of at least three viruses depicted in tables 6-13, preferably using ohgonucleotides comprising at least one primer, probe and/or internal control sequence depicted in a first table of the invention, comprising at least one primer, probe and/or internal control sequence depicted in a second table of the invention, and comprising at least one primer, probe and/or internal control sequence depicted in a third table of the invention or at least one functional equivalent thereof, et cetera.
• a sample is tested in the same assay for the presence of nucleic acid derived from at least two vhus species. More preferably, a sample is tested in the same assay for the presence therein of nucleic acid derived from at least three virus species.
• a nucleic acid amphfication solution is preferably provided with at least part of a sample and with primers of the invention capable of selectively amplifying at least two vhus species.
• Said nucleic acid amplification solution is preferably furthermore provided with probes of the invention specific for said at least two virus species. More preferably, said nucleic acid amphfication solution is furthermore provided with at least one internal control and a probe specific for said internal control.
• ohgonucleotides comprising primer, probe and/or internal control sequences of at least two, preferably at least three, more preferably at least four, most preferably all, tables selected from the group consisting of tables 6-13, or a functional equivalent of at least one of said primer/probe/internal control sequences, are used in a single assay.
• the target viral nucleic acid and the internal control are preferably detected in a multiplex format, e.g. a microarray or fluidic beads system. This way, it is possible to rapidly test a sample for a variety of viruses.
• ohgonucleotides comprising primer, probe and/or internal control sequences of at least two, preferably at least three, most preferably at least tour tables selected from the group consisting of tables 6-13, or a functional equivalent of at least one of said primer/probe/internal control sequences, are used for a real-time PCR in one assay.
• multiplex amplification is only possible for two or at most three different nucleic acid sequences because a plurality of primers, probes and internal controls is at risk of hybridizing with each other instead of their target sequences.
• the primers, probes and internal control sequences of the invention are designed such that ohgonucleotides comprising primer, probe and/or internal control sequences of the invention are suitable for amplification and detection of more than two, preferably more than three, more preferabfy more than four viral nucleic acid sequences simultaneously in the same assay, preferably in a real time PCR.
• This embodiment therefore provides a rapid, sensitive and reliable test for a wide variety of viruses.
• the invention provides an aqueous solution for reverse transcribing an RNA target molecule comprising between 25 and 75 mM of a suitable salt, between 0.05 % and 0.2 % of a non-ionic detergent and a suitable buffer that buffers said solution at a pH between about 8 or 10.
• said solution further comprises between 60 and 240 ⁇ M per nucleotide or equivalent thereof and a reverse transcriptase mentioned above or a functional part, derivative and/or analogue thereof.
• the invention also provides a concentrate of an aqueous solution of the invention. Preferabfy the concentrate comprises between 5 and 10 times the concentration of the chemicals mentioned for the aqueous solution of the invention.
• the invention provides an isolated or recombinant oligonucleotide comprising a sequence as depicted in any one of tables 6-13.
• Said oligonucleotide is suitable for performing a method of the invention.
• a combination of ohgonucleotides comprising at least two sequences depicted in one table selected from tables 6-13 is used. Such combination is particularly suitable for testing a sample for the presence therein of viral nucleic acid.
• a combination of two primers comprising a first primer comprising a sequence of a forward primer depicted in any one of tables 6-13, or a functional equivalent thereof, and a second primer comprising a sequence of the reverse primer depicted in the same table as said forward primer.
• the invention therefore provides a primer pah comprising a primer comprising a sequence of a forward primer as depicted in any one of Tables 6-13, or a functional equivalent thereof, and a primer comprising a sequence of the reverse primer depicted in the same table as said forward primer, or a functional equivalent thereof.
• a preferred primer pair comprises a primer comprising a sequence of a forward primer as depicted in Table 6, or a functional equivalent thereof, and a primer comprising a sequence of the reverse primer depicted in Table 6, or a functional equivalent thereof.
• This primer pah is particularly suitable for testing a sample for the presence of Influenza A.
• Another preferred primer pair comprises a primer comprising a sequence of a forward primer as depicted in Table 11, or a functional equivalent thereof, and a primer comprising a sequence of the reverse primer depicted in Table 11, or a functional equivalent thereof.
• This primer pair is particularfy suitable for testing a sample for the presence of adenovirus.
• Yet another preferred primer pair comprises a primer comprising a sequence of a forward primer as depicted in Table 13, or a functional equivalent thereof, and a primer comprising a sequence of the reverse primer depicted in Table 13, or a functional equivalent thereof.
• This primer pah is particularfy suitable for testing a sample for the presence of enterovirus.
• the invention provides an oligonucleotide comprising a sequence ID#1: 5'-CCC TGA ATG CGG CTA AT-3'; ID#2: 5'-ATT GTC ACC ATA AGC AGC C-3' ; ID#3: EV-specific probe; 5'-GCG GAA CCG ACT ACT TTG GGT- 3'; ID#4: IC-specific probe; 5'-CTT GAG ACG TGC GTG GTA ACC-3' or internal control 5'-CCC TGA ATG CGG CTA ATC CTA ACC ACG GAA CAG GCG GTC GCG AAC CAG TGA CTG GCT TGT CGT AAC GCG CAA GTC TGT GCT TGA GAC GTG CGT GGT AAC CGT CCG TGT TTC CTG TTA TTT TTA TCA TGG CTG CTT ATG GTG ACA AT-3'.
• the invention further comprises a recombinant vhus particle comprising a sequence as depicted in
• the invention further comprises a recombinant vhus particle comprising a control sequence (control probe or the complement thereof).
• said recombinant vhus particle is a recombinant phage particle.
• a phage having, expect for the nucleotide sequence of the region to be amplified using the oligonucleotide of the invention, the characteristics and elements of a phage as described in (24).
• a further embodiment provides a kit for detecting viral nucleic acid comprising at least one and preferably at least two, more preferably at least three ohgonucleotides comprising a sequence as depicted in any one of tables 6-13, or a functional equivalent of said sequence.
• a kit of the invention is particularly suitable for amplifying and/or detecting a virus, in particular any vhus depicted in tables 6-13.
• kits for detecting enteroviral RNA comprising at least one and preferably at least two, more preferabfy at least three ohgonucleotides of the invention.
• Said kit most preferably comprises at least one and preferably at least two, more preferably at least three ohgonucleotides comprising a sequence as depicted in Table 13, or a functional equivalent thereof.
• the kit comprises an internal control nucleic acid of the invention.
• said kit comprises an armoured RNA according to (24) or the example herein, comprising an internal control sequence of the invention, preferably comprising a sequence 5'-CCC TGA ATG CGG CTA ATC CTA ACC ACG GAA CAG GCG GTC GCG AAC CAG TGA CTG GCT TGT CGT AAC GCG CAA GTC TGT GCT TGA GAC GTG CGT GGT AAC CGT CCG TGT TTC CTG TTA TTT TTA TCA TGG CTG CTT ATG GTG ACA AT-3' .
• a kit of the invention comprises ID#4: IC-specific probe; 5'- CTT GAG ACG TGC GTG GTA ACC-3' and/or the complement thereof.
• kits for detecting adenoviral DNA comprising at least one and preferabfy at least two, more preferably at least three ohgonucleotides comprising a sequence as depicted in table 11, or a functional equivalent thereof.
• Said kit preferably comprises an internal control nucleic acid, comprising a sequence as depicted in table 11, or a functional equivalent thereof.
• kits for detecting influenza A RNA comprising at least one and preferably at least two, more preferably at least three ohgonucleotides comprising a sequence as depicted in table 6, or a functional equivalent thereof.
• Said kit preferabfy comprises an internal control nucleic acid, comprising a sequence as depicted in table 6, or a functional equivalent thereof.
• kits of the invention further comprises an aqueous solution of the invention and/or concentrate thereof.
• kit Preferabfy said kit further comprises Superscript II or III.
• lysis buffer, wash buffers, and silica suspension were prepared as described previously (4).
• Superscript II (SS II) was obtained from Life Technologies (Gaithersburg, MD).
• RNAse Inhibitor (RNAsin) was obtained from Promega, Madison, WI).
• Bovine serum albumin (BSA) was obtained from Roche Diagnostics (Almere, The Netherlands).
• Deoxynucleotides dATP, dCTP, dGTP, dUTP
• Taq DNA polymerase Amplitaq Gold
• uracil-N- glycosylase Amperase
• Streptavidin-coated magnetic beads were from Dynal (Hamburg, Germany). Tris, KCl, MgCl 2. Calf Thymus (CT)-DNA, were obtained from Sigma (Zwijndrecht, The Netherlands. Serum and plasma samples were obtained in Vacutamer tubes (Becton Dickinson Systems, Meylan, France). Clinical specimens.
• CPE Cytopathologic effect
• the EV serotypes CVA [1-6, 18-22, 24], CVB [1-6], Echovirus [1-9, 11-22, 24-27, 29-33], Enterovirus [68-71], PV vaccines [1-3]), and Pareehovirus 1 and 2 were kindly provided by the National Institute of Public Health and the Environment (Bilthoven, The Netherlands).
• the rhinovirus serotypes (1A, IB, 3, 8, 11, 13, 14, 15, 16, and 88) were kindly provided by the Department of Virology from the Utrecht Medical Center (Utrecht, The Netherlands).
• the HAV serotype (HM175) was kindly provided by the Municipal Health Service Amsterdam. Primers.
• PCR primers were designed using computer- assisted analysis (OMIGA, Oxford Molecular, England) of all available 5' non- coding regions and full genomes of EV serotypes.
• HPLC-purified PCR primers were from Applied Biosystems (Nieuwerkerk a/d IJssel, The Netherlands) and were diluted in TE buffer to 100 ng/ ⁇ L. Primers for amplification of both wild type EV RNA and IC RNA are located in the conserved 5' non-coding region of EV.
• the primer pair used for amphfication consisted of entero-1 (ID#1: 5'-CCC TGA ATG CGG CTA AT-3'; nucleotide positions [nt] 452-468) and Bio-entero-2 (ID#2: 5'-ATT GTC ACC ATA AGC AGC C-3', 5' biotinylated; nt 579-597).
• Nucleotide numbering was according to the Sabin polio type 2 strain as described by Toyoda et al. (30).
• Armoured EV-RNA control containing part of the 5' non-coding region (nucleotide 428-691) was obtained from Ambion (Ambion, Inc., RNA diagnostics, Austin, Tx, USA) and was constructed according to the armoured RNA technique (24). The method is based on the packaging of recombinant RNA into MS2-like particles, which are produced in Escherichia coli. The particles are isolated through a series of conventional protein purification procedures. According to Ambion, the approximate conversion factor from 1 mg of armoured RNA to copies of RNA is about 2 x 10 14 . The armoured EV-RNA stock solution used in this paper with lot no.
• 040D49012A contained approximately 6.5 x 10 14 EV-RNA c/mL.
• Construction of armoured IC-RNA control For the construction of IC-RNA 2 ohgonucleotides were designed by us and synthesized by Applied Biosystems (Ent-hyb-3; 5'- CCC TGA ATG CGG CTA ATC CTA ACC ACG GAA CAG GCG GTC GCG AAC CAG TGA CTG GTC TGT CGT AAC GCG CAA GTC TGT GCT TGA GAC GTG-3', and Ent-hyb-4; 5' -ATT GTC ACC ATA AGC AGC CAT GAT AAA AAT AAC AGG AAA CAC GGA CGG TTA CCA CGC ACG TCT CAA GCA CAG ACT T-3').
• IC-RNA control was constructed by hybridisation and elongation of 1 ng Ent-byb-3 and 1 ng Ent-hyb-4, 2.5 U of Amplitaq Gold, 5 ⁇ g of BSA, 1 x PCRII buffer (10 mM Tris-HCl; pH 8.3, 50 mM KCl), dATP, dCTP, dGTP, and dTTP at a concentration of 200 ⁇ M each, and 3 mM MgCl 2 . The mixture was incubated for 10 min at 95°C, 5 min at 55°C, and 10 min at 72°C.
• the resulting hybrid was subsequently amplified with primer pair entero-1 and non-Bio-entero-2 in the same mixture as described above and was incubated 10 min at 95°C, followed by 35 cycles each consisting of 20 s at 95°C, 20 s at 55°C, and 1 min at 72 ° C, followed by 5 min at 72°C.
• concentration of resulting amplimer was estimated by measuring the UN-absorption at 260 nm and subsequently 2 ng was cloned into a plasmid vector (PCRII, Promega, Ma) resulting in plasmid pEntIC 2.
• the IC- R ⁇ A sequence was confirmed by dideoxynucleotide sequencing (Visible Genetics Inc., Toronto, Canada).
• Dilution buffer for armoured R ⁇ A controls were diluted in TSM dilution buffer containing 20 ng/ ⁇ L CT-D ⁇ A, 10 mM Tris (pH 7.5), 100 mM NaCl, 1 mM MgCla, 0.1% gelatin (Sigma, catalogue no. (i-9382) and stored at -20°C.
• Final solution of armoured EV-RNA contained 500 copies/ ⁇ L and the final solution of armoured IC-RNA contained 100 copies/ ⁇ L.
• the final reverse transcription mixture (50 ⁇ L) contained 1500 ng hexamers, 1 x CMB1 buffer (10 mM Tris-HCl; pH 8.3, 50 mM KCl, 0.1% Triton), 0.4 U/ ⁇ L of SSII, 120 ⁇ M of each dNTP, 0.08 U/ ⁇ L RNAsin, and 5 mM MgCl 2 .
• the mixture was hicubated for 30 minutes at 42°C and 25 ⁇ L (corresponding to 40 ⁇ L [1/5] of CSF) was subsequently used as input in the PCR.
• the PCR was performed in a 50 ⁇ L volume containing 200 ng of entero-1 (5'-CCC TGA ATG
• PCR's were performed in an Applied Biosystems 9600 thermocycler: 2 min at 50°C, 10 min at 95°C, followed by 45 cj ⁇ cles each consisting of 20 s at 95°C, 20 s at 55°C, and 1 min at 72°C, followed by 5 min at 72°C. Hybridisation and measurement by ECL. After RT-PCR, excess primers were removed as earlier described (6), and amplicons were hybridised with TBR (Tris [2,2'-bipyridine] ruthenium [II] chelate)-labelled probes specific for EV-RNA and IC-RNA as recently described (6).
• TBR Tris [2,2'-bipyridine] ruthenium [II] chelate
• Probes (nt 531-551) were 5' TBR-labelled and were as follows, TBR-entero-1 (ID#3: EV-specific probe; 5'-GCG GAA CCG ACT ACT TTG GGT-3' and TBR-entero-2 (ID#4: IC-specific probe; 5'-CTT GAG ACG TGC GTG GTA ACC-3').
• Hybrids were captured with streptavidin-coated magnetic (beads and the electrochemiluminescence (ECL) signal, expressed in luminosity units (LU), was measured by the M8 system (IGEN, Oxford, England). In real time PCR this step can be omitted. With this device, a 96-well plate is used and unhybridised TBR-labelled probes are automatically removed by washing. The amount of labelled hybrids is determh ed after excitation by applying an electric field.
• a clinical specimen was considered positive for EV-RNA if more than 500 LU were measured with the EV probe, regardless of the result obtained for the IC probe.
• a clinical specimen was considered to be negative for EV-RNA if less than 500 LU were measured with the EV probe and IC- RNA was detected at more than 500 LU.
• 4 controls were included in RNA extraction; 2 positive controls and 2 negative controls.
• the high positive control contained 12,500 armoured EV-RNA copies/extraction and the low positive control (LPC) contained 2500 armoured EV-RNA copies/extraction, both together with 500 armoured IC-RNA copies.
• the first negative control contained 500 armoured IC-RNA copies and served as a control for the entire procedure and should be negative for EV probe but positive for the IC probe.
• the second negative control contained no armoured EV-RNA copies or armoured IC-RNA copies and should be negative for both probes with a mean of 200 LLT.
• Parechovirus specific RT-PCR The conditions of the RT-PCR reaction to detect Parechoviruses were similar as the above described protocol except for the primers used in PCR. For the specific detection of Parechoviruses we used the primer pah as described by Oberste et al. (22).
• Limiting dilutions of the armoured EV-RNA revealed a detection limit of 84 EV-RNA copies in extraction with a 50% hit rate (5/10 runs) resulting in a detection limit of 17 copies of EV-DNA in PCR (1/5).
• Limiting dilutions of the armoured IC-RNA revealed a detection limit of 28 IC-RNA copies in extraction with a 60% hit rate (6/10 runs) resulting in a detection limit of 5 copies of IC- DNA in PCR (1/5) (Table 1). Identical results were found after the direct release of EV-RNA and IC-RNA from its phage (without CSF as background) after incubation at 70°C for 5 minutes (results not shown).
• Isolation of EV in cell culture is still regarded as the diagnostic gold standard.
• There are however many disadvantages to culture such as its labour-intensity, the delay of days to weeks to obtain a positive result, and its false negativity rate of approximately 25-35% because of failures in antibody neutrahzation and the inability of certain CVA serotypes to grow in cell culture (16, 27).
• Many of the disadvantages of cell culture can be overcome by RT-PCR.
• Most frequently used primer and probe sets for the detection of EV are those described by Chapman et al. (10) and Rotbart et al. (27).
• RT-PCR assay in a non-nested format for the detection of EV-RNA in clinical specimens in which 2500 c/mL of armoured IC-RNA mimicking the EV target were included in the RNA extraction and all subsequent steps of the procedure.
• This armoured IC-RNA can be spiked directly into chnicai specimens without degradation of the RNA and enables monitoring of the complete nucleic acid extraction and amplification process for each specimen.
• the sensitivity of the RT-PCR was evaluated with limiting dilutions of both armoured EV-RNA and armoured IC-RNA. We found a detection limit of 5 copies of IC-DNA in 60% of cases in PCR.
• the lower hmit of sensitivity of the RT-PCR was based on the lower detection rate of IC-RNA and was about 150 IC-RNA c/mL.
• the presence of 500 copies of IC-RNA during extraction from 200 ⁇ L of clinical specimens allowed us to draw the conclusion that a specimen found to be negative for EV-RNA but positive for IC-RNA would contain less than 2500 copies of EV-RNA/mL.
• the use of the armoured IC-RNA was critical in the detection of false -negative reactions or invalid results.
• viral culture is still the gold standard to determine an EV infection.
• RT-PCR assays for the diagnosis of EV infections are difficult because cases are defined clinically and viral culture has a poor sensitivity of approximately 70%, partly due to the inability of certain CVA serotj es to grow in cell culture (16, 27).
• CSF provides the most direct link to disease but is usually less successful in virus culture.
• IC-RNA control sequences depicted in Tables 6-12 For the construction of IC-RNA for each virus as hsted m Tables 6-12, two IC- oligonucleotides were designed and synthesized by Applied Biosystems (for sequences of IC-ohgonucleotides, see Tables 6-12). Each set of two ohgonucleotides as given in each of the Tables 6 to 12, contains a stretch of cornplementaiy nucleotides, varying in length between 25 and 50 nucleotides.
• IC- RNA control was constructed by hybridization and elongation of 1 ng of each of the two IC-oligonucleotides (as hsted for each vhus in Tables 6-12), 2.5 U of Amphtaq Gold, 5 ⁇ g of BSA, 1 x PCRII buffer (10 mM Tris-HCl; pH 8.3, 50 mM KCl), dATP, dCTP, dGTP, and dTTP at a concentration of 200 ⁇ M each, and 3 mM MgCl 2 . The mixture was incubated for 10 min at 95°C, 5 min at 55°C, and 10 min at 72°C.
• the resulting hybrid was subsequently amplified with the virus-specific primer pah (listed as forward and reverse in each of the Tables 6-13) in the same mixture as described above and was incubated 10 min at 95°C, followed by 35 cycles each consisting of 20 s at 95°C, 20 s at 55°C, and 1 min at 72°C, followed by 5 min at 72°C.
• concentration of resulting amplicon was estimated by measuring the UV-absorption at 260 nm and subsequently 2 ng was cloned into a plasmid vector (PCRII, Promega, USA). For each virus, the IC-RNA sequence was confirmed by dideoxynucleotide sequencing.
• IC-RNA was made for each virus by in vitro transcription of the IC-containing plasmid with T7 RNA polymerase (Invitrogen, USA). For each vhus the IC-RNA differs from the wild type virus RNA only in the central region of the amplicon, i.e. in the probe region, which allows for discrimination between wild type and IC RNA amplicon after hybridization and detection.
• IC-DNA was made by linearizing the IC-containing plasmid by a unique enzyme, which digests the plasmid outside the amplicon region.
• Adenovirus Materials & methods DNA-purification with MagNApure
• the final PCR mixture (25 ⁇ l) contained: lx Taqman® Universal PCR MasterMix (ABI) 900nM forward primer 900nM reverse primer 200nM wild-type probe 200nM internal control probe 400ng/ ⁇ l ⁇ -casein PCR's were performed in an ABI Prism 7000 sequence detection system as follows:
• the IC DNA was added to the clinical sample in order to monitor extraction and PCR efficiency. Twelve (2-fold) serial dilutions were made from AV DNA and IC DNA in a background of AV negative throat fluid in order to assess the detection limit of the AV PCR. To investigate the linearity, 8 (ten-fold) serial dilutions from AV DNA (with 1000 copies IC DNA in every dilution) in a background of AV negative sputum were tested. A panel of AV prototype strains of all 51 AV serotypes was tested. To establish the chnicai utility of the assay, a comparison of AV PCR and culture was performed in a panel of 152 clinical samples.
• a sensitive AV real-time PCR assay was developed, detecting all 51 AV serotypes.
• the assajr can be applied on different body fluids, among which faeces and respiratory materials.
• the assay has proven to be more rapid and more sensitive than AV culture.
• Primer Adeno-F CAggACgCCTCggAgTACCTgAg
• Primer Adeno-R ggAgCCACCgTggggTT
• Primer of the invention CAggACgCCTCggRgTAYCTSAg Primer of the invention: ggAgCCACVgTgggRTT
• the improved, degenerate primers of the present invention detected all known 51 Ade no viruses.
• Example 5 The improved, degenerate primers of the present invention detected all known 51 Ade no viruses.
• Table 20 shows that over the last year 220 cerebro spinal fluid samples were analyzed for the presence of enterovirus.
• pos means positive for the presence of enterovirus.
• Echovirus 34 is a variant of CVA24.
• Echovirus 8 is a variant of echovirus 1.
• CVA23 is a variant of echovirus 9.
• Echovirus 10 was reclassified as reovirus 1.
• e Echovirus 28 was reclassified as human rhinovirus 1A.
• f Echovirus 22 and 23 were reclassified as parechovhus types 1 and 2.
• Species A type 12, 18, 31
• Cell culture Positive Species B type 3, 7, 11, 14, 16, Cell culture Positive 21, 34, 35, 50
• Species C type 1, 2, 5, 6
• Cell culture Positive Species D type 8-10, 13, 15, 17, Cell culture Positive 19, 20, 22-30, 32, 33, 36-39, 42-49, 51
• Species E type 4
• Cell culture Positive Species F type 40, 41
• Table 17 Comparison of results of Adeno PCR, virus culture, and ELISA with different chnicai specimens from patients with clinically suspected Adeno infections

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• Engineering & Computer Science (AREA)
• Immunology (AREA)
• Wood Science & Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Virology (AREA)
• Biotechnology (AREA)
• Microbiology (AREA)
• Molecular Biology (AREA)
• Physics & Mathematics (AREA)
• Biophysics (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Priority Applications (1)

Applications Claiming Priority (4)

Publications (1)

Family

ID=35456169

Family Applications (1)

Country Status (3)

Families Citing this family (9)

Family Cites Families (7)

• 2005
  • 2005-04-19 EP EP05737536A patent/EP1747296A2/de not_active Withdrawn
  • 2005-04-19 CA CA002563954A patent/CA2563954A1/en not_active Abandoned
  • 2005-04-19 WO PCT/NL2005/000286 patent/WO2005100611A2/en active Application Filing

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events