CA2563954A1 - Detection of viral nucleic acid and method for reverse transcribing rna - Google Patents

Abstract

Provided are means and methods for efficiently reverse transcribing RNA
obtained from a variety of sources. The invention further provides means and methods for efficiently and specifically detecting viruses and particularly human viruses in a sample.

Description

Title: Detection of viral nucleic acid and method for reverse transcribing RNA.
The invention relates to the fields of diagnostics and the copying and/or amplification of nucleic acid. In particular, 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. However, 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.
Human enteroviruses (EV) are members of the family Picornaviridae, are ubiquitous, and are mainly enterically transmitted. Enteroviruses have traditionally been identified by serotype-specific antisera in a virus-neutralizing test and 66 EV types are known to infect humans (20). Sixty-six EV serotypes were initially recognized and divided into 5 major groups: 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), and enteroviruses 68 to 71 (13).
Recent molecular analyses have proven that 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 30 - 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 by 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 may last years before becoming fatal (17).
Early clinical symptoms of meningitis caused by viruses, bacteria, and fungi are quite similar and difficult to distinguish but diagnosis, therapy, and outcome of disease caused by these pathogens vary considerably. Reliable laboratory diagnosis for EV is needed since specific and rapid diagnosis of EV meningitis has a significant impact on patient's management (11). Although, 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.
However, EV can be detected by RT-PCR in all kinds of clinical specimens like whole blood, plasma, sermn, CSF, Stool specimens, throat swabs, vesicle fluids, pleuratic fluids, broncheo-alveolar-lavages, amniotic fluids, urine, and brain biopsies. Early in the acute phase of the illness, EV is frequently isolated from the throat whereas isolation of virus from CSF provides the most direct link to disease but is usually less successful. Cytopathic effect (CPE) is not recognizable within a few days and some enteroviruses do not grow in cell culture and therefore do not cause CPE at all (16, 27).
In recent years, several EV RT-PCR assays have been developed which are sensitive and r apid (9, 10, 15, 27, 28). Although these methods improved the sensitivity and speed of the detection of enteroviruses, it was found that the sensitivity can be further improved by means and methods of the present invention by 40%, range 25-55% (Table 5).
The invention therefore provides a method for reverse transcribing an RNA
target molecule comprising incubating said RNA target molecule with a reverse

2 transcriptase. Preferably, the reverse transcriptase is a Moloney Murine Leukemia Virus Reverse Tr anscriptase [M-MLV RT], more prefer ably a r everse 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, prefer ably 5.0 mM, between 0.05 and 0.2 % of a non-ionic detergent, preferably 0.1%, between 60 and 240 pM per nucleotide, preferably 120 uM 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. Superscript and suitable parts, derivatives and analogues are described in Potter J. et al (2003):
Focus, Vol 25.1, pp 19-24). 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. Preferably 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). Preferably the non-ionic detergent is present in an amount of about 0.1% (vollvol). Preferably, 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. Currently, 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

3 purposes. In general 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.
Preferably, a particular category of nucleotide is present in an amount of between about and 140 uM, more preferably in an amount of about 120 txM. The temperature range is typically a range wherein superscript is active. Prefer ably, 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 pC 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.
Preferably, buffers common in enzymatic processes in molecular biology are used.
Preferably the buffer comprises TRIS or HEPES or a combination or equivalent thereof. Preferably, the solution is buffered by said buffer at a pH of between about 8 and 10. Preferably between about pH 8 and 9. Preferably, at an pH
between about 8.1 and 8.5. More preferably 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. For efficient reverse transcription to take place it is important that the reverse transcriptase primes efficiently.
Efficient priming requires, as yet, the presence of 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. An equivalent of a 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. For random priming, the primer typically contains between about 4 to 10 nucleotides or equivalent thereof.
For specific priming the primer typically contains between about 10 and 30 nucleotides or equivalents thereof, more preferably between about 15 and 25

4 nucleotides, preferably between about 18 and 22 nucleotides, more preferably about 20 nucleotides.
In a particularly preferred embodiment of random priming the primer is a he~amer.. For random priming it is preferred that the primer is a mixture of at least 5 and preferably at least ~0 and more preferably at least 20 oligonucleotides or equivalent thereof, wherein said oligonucleotides or equivalents thereof differ from each other by at least one nucleotide. In a particularly preferred embodiment the primer is a hexamer mirzture comprising at least 20 of the mentioned different oligonucleotides or equivalents thereof.
The RNA may be derived fiom any sample. However, it was found that the incubation conditions for reverse transcribing RNA are more robust than other conditions and allow for efficient reverse transcription of input RNA also when samples are obtained from notoriously difficult sources such as faeces. Thus in a preferred embodiment the invention provides a method of the invention further comprising preparing RNA target molecule from a sample comprising faeces, preferably human faeces.
In one aspect of the invention, the RNA target molecule comprises sequences derived from an enterovirus, preferably a human enterovirus (EV) or an influenza virus, preferably influenza A. Until recently the presence or absence of EV in faeces was analysed using cell lines to propagate any virus fiom the faeces.
Of late, molecular diagnostics have been developed based on for instance amplification of enteroviral nucleic acid. Thus in one embodiment a method of the invention further comprises amplifying produced RT product using one or more primers. In previous studies, EV-RNA was purified without a control (IC-RNA) that monitored both RNA extraction efficiency and RT-PCR efficiency. In molecular diagnostics, the use of an IC-RNA is used for a reliable interpretation of results because it enables the verification of the sensitivity of the assay and avoids false negative results. Thus in one embodiment a method of the invention further comprises providing a sample containing the RNA target molecule vcTith an internal contr of nucleic acid. Prefer ably 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 alieady at an earlier stage, for example to the faeces or the isolated RNA there from. Thus in a preferred embodiment said solution further comprises an internal control RNA molecule. Irz vitro transcribed RNA can be used as an IC-RNA but is prone to degradation by RNAses. , Packaging of IC-RNA into a phage protects the RNA from enzymatic degradation (24). These armoured RNA's can be spiked into clinical specimens without degradation, thus enabling simultaneous monitoring of the complete nucleic acid extraction and amplification process for each specimen. Thus in a preferred embodiment the internal control nucleic acid comprises armoured RNA. In a preferred embodiment 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. Preferably the internal control RNA comprises a sequence 5'-CCC TGA
ATG CGG CTA ATC CTA ACC ACG GAA CAG CTCG GTC GCG AAC CAG TGA
CTCx GCT TCTT CGT AAC C,~CC~ CAA CTTC TCTT GCT TGA CAC C~rTC~ CCTT GCTT
AAC CGT CCG TGT TTC CTCT TTA TTT TTA TCA TGCT CTG CTT ATG GTG
ACA AT-3'. .
Like the wild-type EV target, IC-RNA's should have about the same length, contain the same primer binding sites, and have about the same GC content for ?0 identical extraction and amplification efficiency but should contain a different prol.~e binding site for the differential detection of IC-RNA. Thus in a preferred embodiment the internal control nucleic acid comprises about the same length, primer binding sites) and C-~C content as the region in the EV RNA that is selected for reverse transcription and subsequent amplification. To discriminate between target and internal control 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 betvcTeen internal control and EV-RNA. In a preferred embodiment 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.
Preferably 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).
Such a further conserved region allows the use of one detection means such as a probe for the detection of essentially all (currently known) human enteroviruses.
Thus in one particularly preferred aspect the invention provides a method wherein said internal control RNA molecule comprises a sequence 5'-CCC TGA
ATCT CCTG CTA ATC CTA ACC ACG CTAA CAG GCG C-~TC C~CG AAC CACT TGA
CTCT GCT TGT CGT AAC CTCC~ CAA GTC TGT GCT TGA GAC GTG CC~T CTGT
AAC CGT CCC~ TCTT TTC CTG TTA TTT TTA TCA TGCT CTG CTT ATG GTG
ACA AT-3'. The primers) for amplification 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 easy 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.
Thus in a particularly preferred embodiment 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 TCTA ATCT CGG CTA AT-3' or a functional equivalent thereof and with a second primer comprising a nucleotide sequence (ID#~: 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 l.~ases 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. Accor ding 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. In one embodiment, a functional equivalent of a primer of the W
vention 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 may 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 rever se tr anscr ibing an RNA target molecule according to the invention. In a preferred embodiment a method of the invention further comprises analysing the presence or absence of said nucleic acid by means of a probe. Preferably the presence of nucleic acid derived from a (human) 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. In a preferred embodiment 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 CTTC,~ 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, metapneumovir us, Respiratory Syncitial Vir us, Rhino vir us, Adenovirus and Parainfluenza virus. In these tables, ti, means G or A; S means C-or C and Y means T or C. When at least one primer of tables 6-13 is used for amplification/detection of viral nucleic acid, the sensitivity and/or selectivity of the detection method is improved as compared to conventional methods of the art. According to the present invention, 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. Hence, 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 amplification solution to allow for amplification of said nucleic acid vcrhen present. For instance, if the presence of Influenza A nucleic acid in a sample is investigated, a nucleic acid amplification 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.
Preferably, both primers of any one of tables 6-13, or at least one functional equivalent of at least one of said primers, is used. Hence, if the presence of Influenza A nucleic acid in a sample is investigated, 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. If the presence of Influenza B nucleic acid in a sample is investigated, 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 'l, or at least one functional equivalent of at least one of said primers. lf' the presence of adenovixal nucleic acid in a sample is investigated, 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 oligonucleotide 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. In a preferred embodiment a method of the invention is provided wherein said virus comprises an enterovirus. In a further preferred embodiment 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'-CAGGACC~rCCTCGGRC,~TAYCTSAG-3' or a functional equivalent thereof and said second primer comprises the sequence 5'-GGAGCCACVGTGGGRTT-3' or a functional equivvalent thereof. These primers are particularly suitable for testing a sample for the presence of adenoviral nucleic acid. One further preferred embodiment provides a method of the invention, wherein said first primer comprises the sequence 5'-CTACAAGACCAATCCTCTTCACYTCTG-3' or a functional equivalent thereof and said second primer comprises the sequence 5'-AACTCC-~TCTACGCTGCAC-~TCC-3' or a functional equivalent thereof. These primers are particularly suitable for testing a sample for the presence of influenza A nucleic acid.
In case a sample is tested for the presence of nucleic acid derived from an RNA
virus, said 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.
In a preferred embodiment a method of the invention further comprises analysing the presence or absence of viral nucleic acid by means of a probe.
Preferably the presence or absence of nucleic acid derived from a virus is analysed with a probe comprising a nucleotide sequence of the virus-specific to probe depicted in the same table as said forward primer and/or said reverse primer, or a functional equivalent thereof. Hence, if at least one oligonucleotide comprising a sequence of a primer of Table 6, or a functional equivalent of said primer, is used for amplifying influenza A nucleic acid, a virus-specific probe as depicted in Table G, or a functional equivvalent thereof, is preferably used.
Likewise, if sample nucleic acid is amplified using at least one oligonucleotide comprising a sequence of a primer of another Table (or a functional equivalent thereof), a vir us-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.
In one preferred embodiment, the presence of nucleic acid derived from an enterovirus is analysed with a probe comprising a sequence ID#3: EV-specific probe; 5'-GCC~ GAA CCCT ACT ACT TTG CTCTT-3'or a functional equivalent thereof. In a further preferred embodiment the presence of nucleic acid derived from an adenovirus is analysed with a probe comprising a sequence 5'-CCGGC,TCTC,~GTGCACTTTTCTCCGGC-3'or a functional equivalent thereof. In yet another preferred embodiment the presence of nucleic acid derived from an influenza A virus is analysed with a probe comprising a sequence 5'-TTCACCTCTCACCC,TC~rCCCAGTGAGC-3'or a functional equivalent thereof In order to be capable of verifying the sensitivity of an assay of the invention, and to avoid false negative results, 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. This is for instance performed by adding said internal control to said amplification solution and/or by adding said internal control to at least part of said sample which is used in an amplification reaction. As already explained above, said internal control comprises at least one location where the nucleic acid comprises a sequence that differs from all viruses that are tested for.
Preferably, an internal control depicted in any one of tables G-13 is used. If an oligonucleotide comprising a primer sequence of one specific table (or a functional equivalent thereof) is used, it is preferred to use an oligonucleotide comprising the internal control nucleic acid sequence of the same table (or a functional equivalent thereof). A combination of oligonucleotides 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. As explained above, 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 CTC content for essentially the same extraction and/or amplification efficiency but should contain a differ ent probe binding site for differential detection of inter nal 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.
Preferably, an IC-specific probe comprising a sequence as depicted in Tables 6-is used. The invention thus provides a method of the invention, further comprising analysing the presence or absence of said internal control nucleic acid by 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. Preferably, said probe comprises the sequence 5'-CTT GAG ACG TGC
GTG GTA ACC-3' , 5'-CTATGTGTCCCTCCGTGGTCCCCTGCT-3', and/or 5'-TTCACTGGGCCCCTACTCGCAGTGAC-3', or a functional equivalent thereof, in order to test a sample for the presence of a nucleic acid derived fiom 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. When measured in real time, a probe preferably comprises a fluorophore. Other preferred labels include radio-active and/or digoligenine labels. Label may be associated directly to the probe, or the probe may be detected indirectly, through a labelled bindW g member specific for said probe.
In a further preferred embodiment, 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 G-13. Said two viruses preferably comprise Adenovirus and Influenza.
A method of the invention is preferably performed using at least two oligonucleotides 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 oligonucleotides 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. In one embodiment 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.
More preferably, a sample is tested for the presence of nucleic acid of at least three viruses depicted in tables 6-13, preferably using oligonucleotides 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.
It is desirable to combine as many assays as possible in a single assay format.
This increases the chance of a patient being diagnosed correctly.
In a preferred embodiment, a sample is tested in the same assay for the presence of nucleic acid derived from at least two virus 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. Hence, a nucleic acid amplification solution is preferably provided with at least part of a sample and with primers of the invention capable of selectively amplifying at least two virus 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 amplification solution is furthermore provided with at least one internal control and a probe specific for said internal control.
In a particularly preferred embodiment oligonucleotides 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. In a most preferred embodiment, oligonucleotides comprising primer, probe and/or internal control sequences of at least two, preferably at least three, most preferably at least four 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. Usually, 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. However, the primers, probes and internal control sequences of the invention are designed such that oligonucleotides 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 preferably 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.
In another aspect 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. Preferably said solution further comprises between 60 and 240 uM 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. Preferably the concentrate comprises between 5 and 10 times the concentration of the chemicals mentioned for the aqueous solution of the invention.
In another aspect 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.
Preferably, a combination of oligonucleotides 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.
Preferably, a combination of two primers is provided, comprising a first primer comprising a sequence of a forward primer depicted in any one of tables G-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 pair 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 pair 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 particularly 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 ?0 functional equivalent thereof. This primer pair is particularly suitable for testing a sample for the presence of enterovirus.
In another aspect the invention provides an oligonucleotide comprising a sequence ID#1: 5'-CCC TC-~A 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 GCTT-~5 3'; ID#4: IC-specific probe; 5'-GTT CTAG ACC, TCTC CTTG CTTA ACC-3' or internal control 5'-CCC TGA ATG CGG CTA ATC CTA ACC ACC- CAA CAG CTCG GTC
GCG AAC CACT TGA CTG GCT TGT CCTT AAC GCG CAA GTC TGT GCT TGA
GAC GTG CGT GC~rT AAC CCTT CCCT TGT TTC CTCT TTA TTT TTA TCA TGG
CTG CTT ATG CTTG ACA AT-3'.

The invention further comprises a recombinant virus particle comprising a sequence as depicted in any one of Tables 6-13 or a functional equivalent thereof, or the complement thereof.
The invention further comprises a recombinant virus particle comprising a control sequence (control probe or the complement thereof). Preferably, said recombinant virus particle is a recombinant phage particle. Preferably 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 oligonucleotides 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 virus depicted in tables 6-13.
One preferred embodiment of the invention provides a kit for detecting enteroviral RNA comprising at least one and preferably at least tvvo, more preferably at least three oligonucleotides of the invention. Said kit most preferably comprises at least one and preferably at least two, more preferably at least three oligonucleotides comprising a sequence as depicted in Table 13, or a functional equivalent thereof. Preferably, the kit comprises an internal control nucleic acid of the invention. In one embodiment, said kit comprises an armoured R,NA 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 GCC~ C~rTC GCG AAC CACT TGA CTG
GCT TGT CGT AAC CTCG CAA C~TC TGT GCT TCTA GAC GTG CGT GGT AAC
CGT CCG TC~rT TTC CTG TTA TTT TTA TCA TGG CTG CTT ATG GTG ACA
AT-3' . Preferably, a kit of the invention comprises ID#4: IC-specific probe;
5'-CTT CTAG ACC~r TGC GTG GTA ACC-3' and/or the complement thereof.
1~

Another preferred embodiment of the invention provides a kit for detecting adenovviral DNA comprising at least one and preferably at least two, more preferably at least three oligonucleotides 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.
Yet another preferred embodiment provides a kit for detecting influenza A RNA
comprising at least one and preferably at least two, more preferably at least three oligonucleotides comprising a sequence as depicted in table G, or a functional equivalent thereof. Said kit preferably comprises an internal control nucleic acid, comprising a sequence as depicted in table 6, or a functional equivalent thereof.
In a preferred embodiment a kit of the invention further comprises an aqueous solution of the invention and/or concentrate thereof. Preferably said kit further comprises Super Script II or III.
The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.
Many alternative embodiments can be carried out, which are within the scope of the present invention.

Examples Example 1 Materials and Methods Chemicals and enzymes. 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, dLTTP), Taq DNA polymerase (Amplitaq Gold), uracil-N-glycosylase (Amperase) were from Applied Biosystems (Nieuwerkerk a/d IJssel, The Netherlands). Streptavidin-coated magnetic beads (Dynabeads M-280) were from Dynal (Hamburg, Germany). Tris, KCl, MgCl~, Calf Thymus (CT)-DNA, were ol.~tained from Sigma (Zwijndrecht, The Netherlands. Serum and plasma samples were obtained in Vacutainer tubes (Becton Dickinson Systems, Meylan, France).
Clinical specimens. In total 322 clinical specimens, including 281 CSF
samples, 18 faeces samples, 10 throat swap samples, 3 vesicle fluids, 3 pleuratic fluids, 2 broncheo-alveolar-lavages, 2 amniotic fluids, 1 urine, and 2 brain biopsies, were obtained from patients suspected to be infected with EV and tested by the RT-PCR.
Cytopathologic effect (CPE) for EV infection. Viral culture was done by cocultivation of CSF with human diploid fibroblasts, tertiary monkey kidney cells, or Vero cells. These viral cultures vcTere examined twice weekly for the appearance of EV-specific CPE. Preliminary identification of isolates was performed by either unstained CPE or by CPE incubated with specific Mab (DAKO-Enterovirus, 5-D8/1 [DAIiO, Glostrup, Denmark]).
Serotyping of isolates. Typing of EVs was performed at the National Institute of Public Health and the Environment by neutralization tests with antiserum pools. These pools are prepared according to a combination so designed that an isolate can be screened for identity using 8 pools of 42 anti-sera in a single test.
Viral strains. The EV serotypes CVA [1-G, 18-22, 24], CV'B [1-G], Echovirus [1-9, 11-22, 24-2 7, 29-33], Enterovvirus [68-71], PV vaccines [1-3]), and Parechovirus 1 and 2 were kindly provided by the National Institute of Public Health and the Environment (Bilthoven, The Netherlands). The rhinovirus serotypes (1A, 1B, 3,

8, 11, 13, 14, 15, 1G, 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. Reverse transcription used random hexamers (Roche Diagnostics, Almere, The Netherlands), which were diluted in TE buffer (10 mM Tris-HCI, 1mM EDTA, pH 8.0) to 1.5 ug/gL. 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/uL. 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 amplification consisted of entero-1 (ID#1: 5'-CCC
TGA ATG CGG CTA AT-3'; nucleotide positions [nt] 452-4G8) and Bio-entero-2 (ID#2: 5'-ATT GTC ACC ATA AC-~C ACC 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. The 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 Esch,erichia 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 1014. The armoured EV-RNA stock solution used in this paper with lot no. 040D49012A contained approximately G.5 x 1014 EV-RNA c/mL.

Construction of armoured IC-RNA contr o1. I~'or the construction of 1(.'-.ttNA

oligonucleotides were designed by us and synthesized by Applied Biosystems (Ent-hyb-3; 5'- CCC TGA ATG CGG CTA ATC CTA ACC ACCT CAA CAC GCG
GTC GCG AAC CAC TGA CTG GTC TCTT CC-~T AAC GCG CAA GTC TGT C~rCT
TGA C-~AC C~TC-~-3', and Ent-hyb-4; 5'-ATT GTC ACC ATA ACC ACC CAT CTAT
AAA AAT AAC AGG AAA CAC GGA CGG TTA CCA CGC AGG TCT CAA GCA
CAC ACT T-3'). These 2 oligonucleotides, which together represent the same part of the 5' non-coding region (nucleotide 428-G91) as present in the armoured EV-RNA control, were overlapping over a stretch of 20 nucleotides (underlined) and contained the same primer binding sites as the armoured EV-RNA control (in Itali.es) but with a different probe region (in bold). This IC-probe region allowed for discrimination between EV and IC RNA amplimers after hybridisation and detection. IC-RNA control was constructed by hybridisation and elongation of 1 ng Ent-hyb-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 uM each, and 3 mM MgClz. The mi<Yture was incubated for 10 min at 95°C, 5 min at 55°C, and 10 min at 72°C. The resulting hybr-id 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. The concentration of resulting amplimer was estimated by measuring the UV-absorption at 260 nm and subsequently 2 ng was cloned into a plasmid vector (PCRII, Promega, Ma) resulting in plasmid pEntIC 2. The IC-RNA sequence was confirmed by dideoxynucleotide sequencing (Visible Genetics Inc., Toronto, Canada). Packaging of IC-RNA control into MS2-like particles was performed with plasmid pEntIC 2 as the template and was custom made by Ambion. According to Ambion, the approximate convversion factor from 1 mg of armoured RNA to copies of RNA is about 2 ~ 1014. The armoured IC-RNA stock solution used in this paper with lot no. 021D49013A contained approximately 5.4 x 1014 IC-RNA c/mL.
Dilution buffer for armoured RNA controls. The armoured RNA controls were diluted in TSM dilution buffer containing 20 ng/~L CT-DNA, 10 mM Tris (pH 7.5), 100 mM NaCl, 1 mM MgCl~, 0.1% gelatin (W gma, catalogue no. U~-9332) 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.
RNA purification. EV-RNA was purified from clinical specimens as described earlier (5), with the following modifications; 20 uL of size-fractionated silica particles was used in combination with 900 uL of lysis buffer LG. To this silica-lysis buffer mixture, the clinical specimen and 500 armoured IC-RNA copies were added. RNA was eluted in 100 uL TE buffer.
RT-PCR,. Forty uL of the 100 uL RNA eluate (2/5) was used for reverse transcription. The final reverse transcription milture (50 uL) contained 1500 ng hexamers, 1 x CMB1 buffer (10 mM Tris-HCI; pH 5.3, 50 mM KCl, 0.1% Triton), 0.4 U/uL of SSII, 120 yM of each dNTP, 0.08 U/uL RNAsin, and 5 mM MgCla.
The mixture was W cubated for 30 minutes at 42°C and 25 ~zL
(corresponding to 40 uL [1/5] of CSF) was subsequently used as input in the PCR. The PCR was performed in a 50 yL volume containing 200 ng of entero-1 (5'-CCC TGA ATG
CGG CTA AT-3'; nucleotide positions [nt] 452-468) and 200 ng of Bio-entero-2 (5'-ATT GTC ACC ATA AGC ACTC C-3', 5' biotinylated; nt 579-597), 0.05 U/uL of Amplitaq Cold DNA polymerase, 0.01 U/uL Amperase, 0.1 ug/uL of BSA, 1 x PCRII buffer (10 mM Tris-HCI; pH 8.3, 50 mM KCl), dATP, dCTP, and dGTP at a concentration of 200 uM each, and 400 gM dUTP. The final MgCl~ concentration in PCR reaction was 2.5 mM. PCR's were performed in an Applied Biosystems 9600 thermocycler: 2 min at 50°C, 10 min at 95°C, followed by 45 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.
Hybridisation and measurement by ECL. After RT-PCR, excess primers were removed as earlier described (G), 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 (G). Probes (nt 531-551) were 5' TBR-labelled and were as follows, TBR-entero-1 (ID#3: EV-specific probe; 5'-GCCT GAA CCG
ACT ACT TTG CTGT-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 (LLT), was measured by the M8 system (IC~rEN, 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 determined after excitation by applying an electric field.
Other labels can also be used, such as fluorophores in r eal-time PCT, radioactive labels, digo~igenine etc. A signal of more than 500 LU (= 2.5-times mean background signal for either probe) was considered to be positive.
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. In the diagnostic RT-PCR, 4 controls were included in RNA eitraction; 2 positive controls and 2 negative controls. The high positive control (HPC) contained 12,500 armoured EV-RNA copies/eltraction 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 pair as described by Oberste et al. (22).

Results Determination of the lower limit of detection of the EV RT-PCR assay.
To determine the lower limit of detection of the EV RT-PCR assay, we spiked decreasing amounts of both armoured EV-RNA and armoured IC-RNA diluted in TSM buffer into 200 uL of EV-negative CSF before RNA extraction with the Boom method (5). RNA was diluted in 100 ~L and 40 ~L of extr acted RNA (2/5) was used in RT and subsequently 50% of the cDNA was used in PCR (1/5 of the extracted RNA). 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).
Testing of EV serotypes. All known EV serotypes (n = 64) were tested with the described EV RT-PCR. The extraction of every single EV serotype and non-enterovirus serotype was done alternated with negative extraction controls that contained IC-RNA. All EV serotypes were positive whereas the non-enteroviruses, including echovirus 22 and 23, rhinoviruses, HAV, and all negative controls were negative. The negative results were truly negative since the ECL signals for co-extracted armoured IC-RNA were all positive (Table 2).
Testing of the 2001 and 2002 IaCMD EV proficiency panels. These proficiency panels from 2001 and 2002 consisted of coded freeze-dried samples, including an Echo 11 and CVA 9 dilution series, other EV serotypes representing different genetic clusters of human EV's, and negative controls. The freeze-dried samples were reconstituted with 1 mL of sterile water according to the C~CMD
protocol and 200 pL thereof was used in extraction and amplified as described in the materials and methods section. All samples of the 2001 QCMD proficiency panel were positive regardless of the serotypes and all included negative controls were truly negative, since the ECL signals for co-extracted armoured IC-RNA
were all positive (Table 3).

Testing of clinical specimens. A total of 322 clinical specimens were obtained from patients suspected to be infected with EV and were tested by the RT-PCR.
Forty-five positive results (14%), 267 negative results (82.9%), and 10 invalid results (3.1%) were found. These invalid results were found in 7/281 CSF
samples (2.5%), in 2/18 faeces samples (11.1%), and in 1/10 throat swab samples (10%).
Repeating these 10 invalid samples with the optimised RT-PCR revealed that all invalid results apart from 1 faeces sample were valid, negative for EV, and could be reported to the clinician. (Table 4).
Coinpar ison of virus culture with RT-PCR. Eighty-seven clinical specimens suspected to be infected with EV were available to be tested in virus culture and in RT-PCR. Agreement between virus culture and RT-PCR was found in 72/87 clinical specimens (82.8%). In 54 clinical specimens a negative result was found for both virus culture and RT-PCR, whereas 18 clinical specimens were positive in virus culture and in RT-PCR. Discordant results were found in 15 clinical specimens. In 3 of them virus culture was positive whereas RT-PCR was truly negative for EV-RNA in RT-PCR since the results for IC-RNA were positive.
Tv~To of these RT-PCR negative samples were subjected to a specific Parechovirus RT-PCR and were found to be positive (results not shown). In the other RT-PCR
negative sample CPE was only found after 4 weeks. The remaining discordant results consisted of 12 clinical specimens that were RT-PCR positive and negative in virus culture (Table 5).
2s Discussion.
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 neutralization 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 fiequently used primer and probe sets for the detection of EV are those described by Chapman et al. (10) and Rotbart et al. (27). These primers and probes have been proven to be reactive with all known EV and fail to amplify echovirus 22 and 23, which have been reported to be a genetic distinct genus in the family of Picornaviridae (14, 21, 25). We designed a primer and probe set with minor modifications in comparison to those first described by Chapman et al. and Rotbart et al. and found that not only all known EV serotypes would be detected but also that cross-reactivity between EV serotypes and rhinoviruses was avoided. This cross-reactivity with rhinoviruses has an impact if nasal or pharyngeal swab specimens are used for testing. .
We have described a 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 clinical specimens v~rithout degradation of the RNA and enables monitoring of the complete nucleic acid extr action 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 GO% of cases in PCR. Poisson statistics predicts that 63% of the reactions will be positive with a single copy of DNA in PCR (13). Thus, if RNA
was extracted and reversed transcribed with 100% efficiency, the 60% detection rate should represent 1 copy of IC-DNA in PCR. Since we found comparable results after the direct release of RNA fiom the armoured RNA through incubation at 70°C for 5 minutes and after extraction of the RNA from the armoured RNA using the Boom method, our results might suggest a less efficient reverse transcription step or the presence of a certain percentage of empty armoured RNA phages. In addition, our observed approximately 3-fold difference in detection between EV-RNA and IC-RNA might be explained by inaccuracies in the determination of the concentration and dilution steps of the stock solutions.
The lower limit 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 negativve 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. Currently, viral culture is still the gold standard to determine an EV infection. Validation of a RT-PCR assay for the diagnosis of EV
infections is difficult because cases are defined clinically and viral culture has a poor sensitivity of approximately 70%, partly due to the inability of certain CVA
serotypes to grow in cell culture (16, 27). We found an overall good agreement of 82.8% between virus culture and RT-PCR,. CSF provides the most direct link to disease but is usually less successful in virus culture. We found high discordant results (17.2%) between virus culture and RT-PCR and mainly in CSF samples.
Delayed processing and improper handling of the clinical specimens may have contributed to false negative results in virus culture and our results showed the added value of the described RT-PCR assay for the diagnosis of an EV-infection.
In conclusion, viral EV culture with a good clinical diagnosis will still be of use for EV diagnosis. However, the described EV RT-PCR assay will be of benefit as a sensitive, highly specific, faster assay for the detection of EV in clinical specimens, and the use of an armoured IC RNA strongly reduces false negative results.
Example 2 Construction of IC-RNA control sequences depicted in Tables 6-12.
2~

For the construction of IC-RNA for each virus as listed in 'fables 6-1~, two IC-oligonucleotides were designed and synthesized by Applied Biosystems (for sequences of IC-oligonucleotides, see Tables 6-12). Each set of two oligonucleotides as given in each of the Tables 6 to 12, contains a stretch of complementary 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 listed for each virus in Tables G-12), 2.5 U
of Amplitaq Gold, 5 ~g of BSA, 1 x PCRII buffer (10 mM Tris-HCl; pH 8.3, 50 mM
IiCl), dATP, dCTP, dGTP, and dTTP at a concentration of 200 uM each, and 3 mM MgCI~. 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 pair (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 I2°C, followed by 5 min at 72°C. The 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 i~2 vitro transcription of the IC-containing plasmid with T7 RNA
polymerase (Invitrogen, USA). For each virus 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.
In case of DNA viruses (e.g. adenovirus), IC-DNA was made by linearizing the IC-containing plasmid by a unique enzyme, v~Thich digests the plasmid outside the amplicon region.
Example 3 Adenovvirus: Materials & methods DNA-pu,r°i. fication. wi.tl2 ll~IagNAp~ure 200.1 respiratory specimen (sputum, BAL, etc.) and 350.1 lysisbuffer, or 501 faeces and 500,1 lysisbuffer in an eppendorf tube, vortex.
Pre-lysis 10 min. at room temperature.
Spin for 2 min at maximum speed (13000 rpm).
Transfer 490.1 supernatant to a MagNApure sample cartridge.
Add 10.000 copies of Adeno internal control (101 with a concentration of 10E6 cop./ml) MagNApure total NA isolation-external lysis protocol, elution volume 100w1.
10y1 of each eluate was used for Taqman PCR (10.1 contains 1000 copies of internal control).
The final PCR mixture (25 ~.1) contained:
lx TaqmanC> Universal PCR MasterMix (ABI) 900nM forward primer 900nM reverse primer 200nM wild-type probe 200nM internal control probe 400ng/~l a-casein PCR's were performed in an ABI Prism 7000 sequence detection system as follows:
2 min 50°C and 10 min at 95°C, followed by 45 cycles each consisting of 15 sec at 95°C and 1 min at 60°C.
Real-time PCR with an internal control detecting all 51 known Adenovirus serotypes Objectives: The gold standard for the diagnosis of adenovirus (A~ infection is culture. However, results are available after days to weeks and not all AV
types grow in culture. Especially in immune compromised patients severe mtections with AV are described and a rapid diagnosis would be important. Therefore, an AV real-time PCR was developed, detecting all 51 known AV serotypes.
Methods: Primers were chosen in the hexon region. In order to cover all 51 serotypes the forward primer is degenerated at 3 positions and the reverse primer at 2 positions. The IC DNA contains the same primer binding sites, but has a shuffled probe region compared to WT virus. Primers, probes and internal control are depicted in Table 11. The IC DNA was added to the clinical sample in order to monitor extraction and PCR efCciency. 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 str ains of all 51 AV serot5~aes was tested. To establish the clinical utility of the assay, a comparison of AV PCR and culture was performed in a panel of 152 clinical samples.
Results: In the serial dilutions in throat fluid the 50% detection limit of AV
DNA
was 8 copies per PCR assay (400 copies/ml) and of IC DNA 16 copies per PCR, assay (800 copies/ml) (Table 14). The real-time AV PCR was linear from 125 copies per PCR assay (6250 copies/ml) until 1.25 E8 copies per PCR assay (G.25 E9 copies/ml) (Table 15). All 51 AV serotypes were detected in the panel of AV
prototype strains (Table 18). Concordant results between culture or Ag detection and PCR v~~ere found in 139/152 (91.4%). In 10 cases, PCR was positive while culture was negative (G.6%) and in 12 cases PCR was positive while both culture and ELISA were negative (7.7%). In 1 case, PCR was negative while culture was positive (0.6%)(Table 17).
Conclusion: A sensitive AV real-time PCR assay was developed, detecting all 51 AV serotypes. The assay 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.

Example 4 Primers of the invention with degenerated sites (Table 11) were compared with primers without degenerated sites in an Adenovirus detection experiment.
The results are shown in Table 18.
Left part of Table 1~ shows results obtained with primers Adeno-F and Adeno-R
without degenerated sites:
Primer Adeno-F: CAggACgCCTCggAgTACCTbAg Primer Adeno-R: ggAgCCACCgTggggTT
Right part of Table 1S shows results obtained with primers of the present invention with degenerated sites:
Primer of the invention: CAggACgCCTCggRgTAYGTSAg Primer of the invention: ggAgCCACVgTgggRTT .-~0 Positive results are defined by detectable adenovirus CT value regardless of IC
values. Negative Adenovirus results are true negative results only if IC is detectable as is indicated by CT values.
Results:
~5 The non-degenerate primers failed to detect 4 Adenovirus serotypes.
The improved, degenerate primers of the present invention detected all known Adenoviruses.

Example 5 Primers of the invention for detecting enterovirus were used in the following experiment:
From a previous series five samples that were cultured and found to represent an enterovir us infection (based on CPE), were tested at the AMC, using the newly developed AMC primer and probe set. The followed method is the same as described in the Material and Methods section of Example 1. All five samples were found to be positive for enterovirus (subtypes Echo 9, Echo 18, COX B5, COX A9, and EV Stuttgart).
This shows that the optimized AMC primer and probe set is capable of detecting various enteroviral subtypes.
Example 6 Over the last twelve months a large series of cerebro spinal fluid samples from patients suspected to suffer fiom myocarditis, sepsis, diarrhea, meningitis and respiratory disorders was analyzed at the Dept. of Clinical Virology. The samples were analyzed by PCR (see Materials and Methods section of Example 1).
Table 20 shovcTs that over the last year 220 cerebro spinal fluid samples were analyzed for the presence of enterovirus. In the column 'Test result', pos means positive for the presence of enterovirus.
It is concluded from Table 20 that of the 220 cerebro spinal fluid samples that were analyzed at the AMC over the last year, 32 were positive. Hence, in a significant amount of cases enterovirus is detected. The AMC primer and probe set is therefore a significant improvement in the diagnostics of viruses in patient samples.

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W. Doerr. 1997. Rapid diagnosis of enterovirus infection by a new one-step reverse transcription-PCR assay. J.CIin.Microbiol. 35:976-977.

13. Lipson, S. M., R. Walderman, P. Costello, and K. Szabo. 1988.
Sensitivity of rhabdomyosarcoma and guinea pig embryo cell cultures to field isolates of difficult-to-cultivate group A Co~sackieviruses.
J.Clin.Microbiol. 26:1298-1303.

14. Mcliinney, R. E., Jr., S. L. Katz, and C. M. Wilfert. 1987. Chronic enteroviral meningoencephalitis in agammaglobulinemic patients.
Rev.Infect.Dis. 9:334-356.

15. Melnick, J. L. 1996. Enteroviruses: Polioviruses, Co~sackieviruses, echoviruses, and newer enteroviruses., p. 655-712. In D. M. IW ipe and P. M. Howley (eds.), Fields. Lippincott-Raven, Philadelphia.

16. Modlin, J. F., R. Daban, L. E. Berlin, D. M. Virshup, R. H.
Yolken, and M. Menegus. 1991. Focal encephalitis with enterovirus infections. Pediatrics 58:841-845.

17. Muir, P., U. Kammerer, K. Korn, M. N. Mulders, T. Poyry, B.
Weissbrich, R. Kandolf, G. M. Cleator, and A. M. van Loon. 199S.
Molecular typing of enteroviruses: current status and future requirements. The European Union Concerted Action on Virus Meningitis and Encephalitis. Clin.Microbiol.Rev. 11:202-227.

18. Oberste, M. S., K. Maker, and M. A. Pallansch. 1998. Complete sequence of echovirus 23 and its relationship to echovirus 22 and other human enteroviruses. Virus Res. 56:217-223.

19. Oberste, M. S., K. Maker, and M. A. Pallansch. 1999. Specific detection of echoviruses 22 and 23 in cell culture supernatants by RT-PCR. J.Med.Virol. 58:178-181.

20. Pallansch, M. A. and R. P. Roos. 2001. .~nterovi.ruses: polioviruses, Coxsackieviruses, echoviruses and newer enteroviruses, p. 723-776. Ifz D. M. Knipe and P. M. Howley (eds.), Fields. Lippicott Williams R
Wilkins, Philadelphia.

21. Pasloske, B. L., C. R. Walker peach, R. D. Obermoeller, M.
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23. Rotbar t, H. A., M. H. Sawyer, S. Fast, C. Lewinski, N. Murphy, E.
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Diagnosis of enteroviral meningitis by using PCR with a colorimetric microwell detection assay. J.Clin.Microbiol. 32:2590-2592.

24. Rotbart, H. A. 1995. Enteroviral infections of the central nervous system. Clin.Infect.Dis. 20:971-981.

25. Stellrecht, K. A., I. Harding, F. M. Hussain, N. G. Mishrik, R. T.
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Imura, and A. Nomoto. 1984. Complete nucleotide sequences of all three poliovirus serotype genomes. Implication for genetic relationship, gene function and antigenic determinants. J.Mol.Biol. 174:561-585.

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Table 1 a. Sensitivity for armored EV-RNA control pnages.
EV-RNA copies/200 ~L
CSF in extraction in RT (2/5) in PCR (1/5)* proportion positive 900 360 180 10/10 (100%) 450 180 90 9/10 (90%) 225 90 45 8/10 (80%) 11~ 45 23 8/10 (80%) 84 34 17 5/10 (50%) 56 23 11 6/20 (30%) 28 11 6 5/16 (31%) 14 6 3 1/10 (10%) 7 3 1 1/10 (10%) 0 0 0 0/10 (0%) *For the copy numbers presented in Table 1 the following assumptions were made; (l) The armoured RNA stocks contain the specified number of phage particles and all contain EV-RNA or IC-RNA, (ii) a 100% efficiency in extraction, in r ever se tr anscription, and in PCR.

Table 1b. Sensitivity for armoured IC-RNA control phages.
IC-RNA copies/200 wL
CSF in e~tr action in RT (2/5) in PCR (1/5)* proportion positive 225 90 45 10/10 (100%) 112 45 23 9/10 (90%) 56 23 11 8/10 (SO%) 28 11 G G/10 (GO%) 0 0 0 0/10 (0%) For the copy numbers presented in Table 1 the following assumptions were made; (i) The armoured R.NA stocks contain the specified number of phage particles and all contain EV-RNA or IC-RNA, (ii) a 100% efficiency in extraction, in reverse transcription, and in PCR. _ Table 2. specificity of EV RT-PCR for protot5~ae strata s of enteroviruses and controls.
Virus serotype Source Result Mean LU EV-RNA (range) CVA (2-4, G-18, 20, Cell culturePos 87,000 (57,000-117,000) 21, 24a) CVA (1, 5, 19, 22) Suckling Pos 87,000 (73,000-100,000) mice CVB (1- 6) Cell culturePos 91,000 (75,000-107,000) Echovirus (1-9, 11-1G,Cell culturePos 98,000 (86,000-111,000) 21, 24-27, 29-33 b~~~~,e) Echo~~irus (22, 23~ Cell cultureNeg 218 (209-227) Enterovirus (G8-71) Cell culturePos 77,000 (68,000-86,000) Poliovirus (1, 2, Cell culturePos 104,000 (79,000-129,000) 3) Rhinovirus (1A, 1B, Cell cultureNeg 228 (205-277) 3, 8, 11, 13, 14, 15, 1G, 88) HAV Cell cultureNeg 237 Negative control Neg 226 (217-235) HPC (25,000 EV-RNA Pos 85,000 (62,000-106,000) c/mL) LPC (5,000 EV-RNA Pos 58,000 (46,000-70,000) c/mL) IC (2,500 IC RNA c/mL) Pos 51,000 (40,000-63,0001 a Echovirus 34 is a variant of CVA24.
~ Echovirus 8 is a variant of echovirus 1.
CVA23 is a variant of echovirus 9.
d Echovirus 10 was reclassified as reovirus 1.
a Echovirus 28 was reclassified as human rhinovirus 1A.
f Echovirus 22 and 23 were reclassified as parechovirus types 1 and 2.
Source: adapted from Melnick, Fields Virology, 3rd edition, 1996 (18).

Table 3. Composition and results of the QCMD proficiency panel 2UU 1.
Virus LU EV- LU IC- Result Code ~erotype titer * RNA RNA
EV-CO1 CXVA 9 0.036 31,104 65,482 Positive EV-C02 CXVA 9 0.36 87,325 81,793 Positive EV-C03 CYVA 9 3.6 90,570 71,435 Positive EV-C04 No virus 214 89,860 Negative EV-C06 Echo 11 25.2 96,460 85,396 Positive EV-C11 Echo 11 252 99,930 43,751 Positive EV-C09 Echo 11 25,200 87,380 860 Positive EV-C07 CXVB 5 317 83,867 32,092 Positive EV-C08 EV 71 56.4 98,508 31,411 Positive EV-C10 No virus 226 74,234 Negative EV-C05 Echo G 20,000 82,529 27,549 Positive *Titer of original virus stocks before inactivation and fieeze-drying (TCIDso/mL):
CVA 9; 3.G x 106, Echo G; 2.0 x 108, Echo 11; 2.5 x 10~, CVB 5; 3.2 x 10~, EV
71;
5.6 x 10~.

Table 4. Summarv of results after testing clinical specimens.
Specimen N Positive Negative Faeces 18 G 10 Throat swabs 10 0 9 Other 13 3 10 Total no. 322 45 (14%) 2G7 (82.9%) Table 5. Comparison of RT-PCR and virus culture on different clinical specimens obtained from patients with a clinically suspected EV infection.
Clinical specimen No tested RT-PCR + RT-PCR + RT-PCR - RT-PCR -Culture + Culture - Culture - Culture +

Faeces 13 3 1 9 0 Throat swap 4 0 0 4 0 Urine 1 0 0 1 0 Biopsy 1 0 1 0 0 Total no. 86 18 (20.9%) 12 (14.0%)54 (62.8%)2* (2.3%) * These culture isolates were identified as Parechovirus and not as Enterovirus.

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Table 14. PCR sensitivity for armoured AV DNA and IC DNA control phages DNA type and no. of No. of DNA copies Proportion positive Copies/200u1 of throat swab in the PCR mi..iture (1/10) AV DNA
2500 250 12/12 (100%) 1250 125 12/12 (100%) 620 62 12/12 (100%) 310 31 10/12 (83,3%) 160 16 10/12 (83,3%) 80 8 6/12 (50%) 40 4 1/12 (8,3%) 0 0 0/12 (0%) IC DNA
2500 250 12/12 (100%) 1250 125 12/12 (100%) 620 G2 11/12 (91,7%) 310 31 10/12 (83,3%) 1G0 16 5/12 (50%) ...

80 8 3/12 (25%) 40 4 2/13 (15,4%) 0 0 0/11 (0%) Table 15. Linearity of AV Taqman PCR
No. of AV DNA No. of AV DNA copies Proportion positive Copies/200u1 of sputuma in the PCR mixture (1/10)b 1,25 E9 1,25 E8 8/8 1,25 E8 1,25 E7 8/8 1,25 E7 1,25 E6 8/8 1,25 EG 1,25 E5 8/8 1,25 E5 1,25 E4 S/8 1,25 E4 1250 8/8 125 12,5 5/8 a In each sample 10.000 copies of IC DNA per 200 gl. of sputum.
b In each PCR mixture 1000 copies of IC DNA
Linearity standard curve: exclusive 12,5 copies input slope= -3,35 and R~=0,998; inclusive 12,5 copies input slope= -3,3S and R2=0,990 Table 16. Sensitivity and specificity of AV PCR for prototype strains of AVs and contr ols Virus Sour ce Result Species A (type 12, 18, Cell culture Positive 31) Species B (type 3, 'l, 11, Cell culture Positive 14, 16, ?1, 34, 35, 50) Species C (type 1, 2, 5, Cell culture Positive 6) Species D (type 8-10, 13, Cell culture Positive 15, 17, 19, 20, 22-30, 32, 33, 36-39, 42-49, 51) Species E (type 4) Cell culture Positive Species F (type 40, 41) Cell culture Positive Other virus Enterovirus Negative Negative control Calf-thymus DNA Negative HPC Plasmid DNA Positive LPC Plasmid DNA Positive IC Plasmid DNA Positive U
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Table 19~
AMC primer and probe sequences Primer Sequence Position C Entero 1 5'-CCC.TgA.Atg.Cgg.CTA.AT- 3' (452 -468)#

C Entero ~ 5'-ATT.gTC.ACC.ATA.AgC.AgC.C- (579 -3' 597)#

C wildtype probe5'-gCg.gAA.CCg.ACT.ACT.TTg.ggT- (531- 551)#
3' C IC probe 5'-CTT.gAg.AC .TgC.gTg.gTA.ACC- (531- 551)#
3' # = numbering according to Toyoda et al. 1984.

Table 20 Sample Sampling Material Test Date Result 20471597O1-04-2004CEREBRO SPINAL FLLTIDPos 2047191821-04-2004CEREBRO SPINAL FLLTIDPos 2047218610-05-2004CEREBRO SPINAL FLUID Pos 2047224212-05-2004CEREBRO SPINAL FLLTIDPos 2047271607-06-2004CEREBRO SPINAL FLUID Pos 2047295120-06-2004CEREBRO SPINAL FLUID Pos 2047314924-06-2004CEREBRO SPINAL FLUID Pos 2047314725-06-2004CEREBRO SPINAL FLLTIDPos 2047362628-07-2004CEREBRO SPINAL FLUID Pos 2047367502-OS-2004CEREBRO SPINAL FLLTIDPos 2047373203-OS-2004CEREBRO SPINAL FLUID Pos 2047383809-08-2004CEREBRO SPINAL FLUID Pos 2047390913-OS-2004CEREBRO SPINAL FLUID Pos 2047390313-OS-2004CEREBRO SPINAL FLUID Pos 2047403523-08-2004CEREBRO SPINAL FLUID Pos 2047421925-OS-2004CEREBRO SPINAL FLUID Pos 2047429503-09-2004CEREBRO SPINAL FLUID Pos 2047445509-09-2004CEREBRO SPINAL FLUID Pos 2047506227-09-2004CEREBRO SPINAL FLUID Pos 2047473629-09-2004CEREBRO SPINAL FLUID Pos 2047496412-10-2004CEREBRO SPINAL FLUID Pos 2047500712-10-2004CEREBRO SPINAL FLUID Pos 2047512515-10-2004CEREBRO SPINAL FLUID Pos 2047515520-10-2004CEREBRO SPINAL FLUID Pos 2047524926-10-2004CEREBRO SPINAL FLUID Pos 2047551509-11-2004CEREBRO SPINAL FLUID Pos 2047551509-11-2004CEREBRO SPINAL FLUID Pos 2047569018-11-2004CEREBRO SPINAL FLUID Pos 20570105O1-O1-2005CEREBRO SPINAL FLUID Pos 2057067102-02-2005CEREBRO SPINAL FLUID Pos 2057102721-02-2005CEREBRO SPINAL FLUID Pos 2057131405-03-2005CEREBRO SPINAL FLUID Pos 2047161803-04-2004CEREBRO SPINAL FLUID Ne 2047166405-04-2004CEREBRO SPINAL FLUID Neg 2045075405-04-2004CEREBRO SPINAL FLUID Ne 2047169006-04-2004CEREBRO SPINAL FLUID Ne 2047173206-04-2004CEREBRO SPINAL FLUID Neg 2047169207-04-2004CEREBRO SPINAL FLUID Ne 20471770OS-04-2004CEREBRO SPINAL FLUID Ne 2047172008-04-2004CEREBRO SPINAL FLUID Ne 2047173409-04-2004CEREBRO SPINAL FLLTIDNe 2047176214-04-2004CEREBRO SPINAL FLUID Neg 2047176614-04-2004CEREBRO SPINAL FLUID Ne 2047176814-04-2004CEREBRO SPINAL FLUID Ne 2047184016-04-2004CEREBRO SPINAL FLUID Neg 2047153818-04-2004CEREBRO SPINAL FLUID Ne 2047184118-04-2004CEREBRO SPINAL FLUID Neg ~ ~ ~

2047183919-04-2004CEREBRO SPINAL FLUID Ne 2047187620-04-2004CEREBRO SPINAL FLUID Ne 2047194321-04-2004CEREBRO SPINAL FLUID Ne 2047189121-04-004CEREBRO SPINAL FLUID Ne 2047191921-04-2004GEREBRO SPINAL FLUID Ne 2047209029-04-2004CEREBRO SPINAL FLUID Ne 2047207703-05-2004GEREBRO SPINAL FLUID Ne 2047211504-05-2004CEREBRO SPINAL FLUID Ne 2045098404-05-2004CEREBRO SPINAL FLUID Ne 2047219905-05-2004CEREBRO SPINAL FLUID Ne 2047211405-05-2004CEREBRO SPINAL FLUID Ne 2047220306-05-2004CEREBRO SPINAL FLUID Ne 2047218408-05-2004GEREBRO SPINAL FLUID Ne 2047233013-05-2004CEREBRO SPINAL FLUID Ne 2047232314-05-2004CEREBRO SPINAL FLLTIDNe 2047232214-05-2004GEREBRO SPINAL FLUID Ne 2047237717-05-2004CEREBRO SPINAL FLUID Neg 2047235417-05-2004CEREBRO SPINAL FLUID Ne 2047234418-05-2004GEREBRO SPINAL FLUID Ne 2047248521-05-2004CEREBRO SPINAL FLUID Neg 2047243123-05-2004CEREBRO SPINAL FLUID Ne 2047250827-05-2004CEREBRO SPINAL FLUID Neg 2047253428-05-2004GEREBRO SPINAL FLUID Ne 2047253928-05-2004GEREBRO SPINAL FLUID Ne 2047253730-05-2004CEREBRO SPINAL FLUID Neg 2047253330-05-2004CEREBRO SPINAL FLUID Ne 2047258130-05-2004CEREBRO SPINAL FLUID Neg 2047254031-05-2004CEREBRO SPINAL FLUID Ne 2047267531-05-2004CEREBRO SPINAL FLUID Ne 2047267631-05-2004CEREBRO SPINAL FLUID Ne 2047267402-06-2004CEREBRO SPINAL FLLTIDNe 2047271505-06-2004GEREBRO SPINAL FLUID Ne 2047267006-OG-2004CEREBRO SPINAL FLUID Ne 2047267307-06-2004CEREBRO SPINAL FLUID Ne 2047270508-OG-2004CEREBRO SPINAL FLUID Ne 204775510-06-2004CEREBRO SPINAL FLUID Ne 2047280911-06-2004CEREBRO SPINAL FLUID Ne 2047281112-OG-2004CEREBRO SPINAL FLUID Ne 2047280814-OG-2004CEREBRO SPINAL FLUID Ne 2047285615-06-2004CEREBRO SPINAL FLUID Neg 2047285715-06-2004CEREBRO SPINAL FLUID Ne 2047285815-06-2004GEREBRO SPINAL FLUID Ne 2047296316-06-2004CEREBRO SPINAL FLUID Ne 2047285916-06-2004GEREBRO SPINAL FLUID Ne 2047289917-OG-2004CEREBRO SPINAL FLUID Ne 2047296418-06-2004CEREBRO SPINAL FLUID Ne 2047296021-06-2004CEREBRO SPINAL FLUID Ne 2047303022-06-2004GEREBRO SPINAL FLUTD Ne 2047299822-06-2004CEREBRO SPINAL FLUID Ne 2047299923-06-2004CEREBRO SPINAL FLUID Ne 2047309323-OG-2004CEREBRO SPINAL FLUTD Ne 2047297823-06-2004CEREBRO SPINAL FLUID Ne 2047299723-06-2004CEREBRO SPINAL FLUID Ne 2047309523-06-2004CEREBRO SPINAL FLUID Ne 2047308927-06-2004CEREBRO SPINAL FLUID Neg 2047317528-OG-2004CEREBRO SPINAL FLLTID Ne 2047309128-06-2004CEREBRO SPINAL FLUID Ne 2047364429-OG-2004CEREBRO SPINAL FLLTID Neg 2047313329-06-2004CEREBRO SPINAL FLUID Ne 2047312830-06-2004CEREBRO SPINAL FLUID Ne 20473168O1-07-2004CEREBRO SPINAL FLUID Ne 20473169O1-07-2004CEREBRO SPINAL FLUID Ne 2047331803-07-2004CEREBRO SPINAL FLUID Neg 2047330104-07-2004CEREBRO SPINAL FLUID Ne 2047323004-07-2004CEREBRO SPINAL FLUID Neg 2047336309-07-2004CEREBRO SPINAL FLUID Ne 2047337109-07-2004CEREBRO SPINAL FLUID Ne 2047335909-07-2004CEREBRO SPINAL FLUID Ne 2047336511-07-2004CEREBRO SPINAL FLUID Ne 2047335711-07-2004CEREBRO SPINAL FLUID Ne 2047336612-07-2004GEREBRO SPINAL FLUID Neg 2047340013-07-2004GEREBRO SPINAL FLUID Ne 204734701G-07-2004CEREBRO SPINAL FLUID Ne 2047348217-07-2004CEREBRO SPINAL FLUID Ne 2047347118-07-2004CEREBRO SPINAL FLUID Ne 2047348519-07-2004CEREBRO SPINAL FLUID Neg 2047352320-07-2004CEREBRO SPINAL FLUID Ne 2047358921-07-2004CEREBRO SPINAL FLUID Ne 2047352021-07-2004CEREBRO SPINAL FLLTID Ne 2047354822-07-2004CEREBR.O SPINAL FLUID Ne 2047359423-07-2004GEREBRO SPINAL FLUID Neg 2047354923-07-2004CEREBRO SPINAL FLUID Ne 204735992G-07-2004CEREBRO SPINAL FLUID Ne 204735902G-07-2004CEREBRO SPINAL FLUID Ne 2047362826-07-2004GEREBRO SPINAL FLUID Ne 2047359827-07-2004CEREBR.O SPINAL FLUID Ne 2047363128-07-2004CEREBRO SPINAL FLUID Ne 2047362728-07-2004CEREBRO SPINAL FLUID Ne 2047368631-07-2004CEREBRO SPINAL FLUID Ne 2047372231-07-2004CEREBRO SPINAL FLUID Ne 2047368502-O8-2004CEREBRO SPINAL FLUID Ne 2047368102-08-2004CEREBRO SPINAL FLUID Ne 2047373103-08-2004CEREBRO SPINAL FLUID Ne 2047373303-08-2004GEREBRO SPINAL FLUID Ne 2047372303-08-2004CEREBRO SPINAL FLLTID Ne 2047373504-08-2004CEREBRO SPINAL FLUID Neg 2047379706-08-2004GEREBRO SPINAL FLUID Ne 2047383707-08-2004CEREBRO SPINAL FLUID Ne 2047379908-08-2004CEREBRO SPINAL FLUID Neg 2047380309-08-2004CEREBRO SPINAL FLLTTD Ne _2047380209-08-2004GEREBRO SPTNAL FLUID Ne 2047383911-08-2004CEREBRO SPTNAL FLUID Ne 2047394817-08-2004CEREBRO SPINAL FLUID Ne 2047394018-08-2004CEREBRO SPINAL FLUID Ne 2047394118-08-2004CEREBRO SPINAL FLUID Ne 2047404819-08-2004CEREBRO SPINAL FLUID Ne 2047404921-08-2004CEREBRO SPINAL FLUID Ne 2047404123-08-2004CEREBRO SPINAL FLUID Ne 2047409424-08-2004CEREBRO SPINAL FLUID Ne 2047408924-OS-2004CEREBRO SPINAL FLUID Ne 2047408125-08-2004CEREBRO SPINAL FLUID Neg 2047409325-08-2004CEREBRO SPINAL FLUID Ne 2047421829-OS-2004CEREBRO SPINAL FLUID Ne 2047417430-08-2004CEREBRO SPINAL FLUID Ne 2047421331-08-2004CEREBRO SPINAL FLLTIDNe 2047421231-08-2004CEREBRO SPINAL FLUID Neg 2047460501-09-2004CEREBRO SPINAL FLUID Ne 2047421101-09-2004CEREBRO SPINAL FLUID Ne 2047458002-09-2004CEREBRO SPINAL FLUID Ne 2047430003-09-2004CEREBRO SPINAL FLUID Ne 2047430203-09-2004CEREBRO SPINAL FLUID Ne 2047430404-09-2004GEREBRO SPINAL FLUID Ne 2047460705-09-2004CEREBRO SPINAL FLLTIDNe 2047433906-09-2004CEREBRO SPINAL FLUID Nea 2047433507-09-2004CEREBR.O SPINAL FLUIDNe 2047433707-09-2004CEREBRO SPINAL FLUID Ne 2047433308-09-2004CEREBRO SPINAL FLUID Neg 2047435810-09-2004CEREBRO SPINAL FLUID Ne 2047444910-09-2004CEREBRO SPINAL FLUID Ne 2047440511-09-2004CEREBRO SPINAL FLLTIDNe 2047440412-09-2004CEREBRO SPINAL FLUID Ne 2047441012-09-2004CEREBRO SPINAL FLUID Ne 2047444814-09-2004CEREBRO SPINAL FLUID Ne 2047443715-09-2004CEREBRO SPINAL FLUID Neg 2047443615-09-2004CEREBRO SPINAL FLUID Ne 204744791G-09-2004CEREBRO SPINAL FLLTIDNe 2047453116-09-2004CEREBRO SPINAL FLUID Ne 204744761G-09-2004CEREBRO SPINAL FLUID Ne 2047452420-09-2004CEREBRO SPINAL FLUID Ne 2047457921-09-2004CEREBRO SPINAL FLUID Ne 2047457721-09-2004CEREBRO SPINAL FLUID Ne 2047457821-09-2004CEREBRO SPINAL FLUID Neg 2047460822-09-2004CEREBRO SPINAL FLUID Ne 2047461423-09-2004CEREBRO SPINAL FLLTIDNe 2047466023-09-2004CEREBRO SPINAL FLUID Ne 2047460623-09-2004CEREBRO SPINAL FLUID Ne 2047460923-09-2004CEREBRO SPINAL FLUID Ne 2047465624-09-2004CEREBRO SPINAL FLUID Ne 2047464924-09-2004CEREBRO SPINAL FLUID Ne 2047464825-09-2004CEREBRO SPINAL FLUID Neg 20474740O1-10-2004CEREBRO SPINAL FLUID Ne 2047477803-10-2004CEREBRO SPINAL FLLTIDNe 2047478304-10-2004CEREBRO SPINAL FLUID Neg 2047482206-10-2004CEREBRO SPINAL FLUID Ne 2047490610-10-004CEREBRO SPINAL FLUID Neg 2047496311-10-2004GEREBRO SPINAL FLUID Ne 2047491211-10-2004CEREBRO SPINAL FLUID Ne 2047496613-10-2004CEREBRO SPINAL FLUID Ne 2047500214-10-2004CEREBRO SPINAL FLUID Ne 2047519023-10-2004CEREBRO SPINAL FLUID Neg 2047519325-10-2004CEREBRO SPINAL FLUID Ne 2047524826-10-2004CEREBRO SPINAL FLUID Neg 2047529227-10-2004CEREBRO SPINAL FLUID Ne 2047533429-10-2004GEREBRO SPINAL FLUID Ne 20475333O1-11-2004CEREBRO SPINAL FLUID Ne 2047541803-11-2004CEREBRO SPINAL FLUID Ne 2047546503-11-2004CEREBRO SPINAL FLUID Ne 2047550908-11-2004CEREBRO SPINAL FLUID Ne 2047553911-11-2004CEREBRO SPINAL FLUID Ne 2045238611-11-2004CEREBRO SPINAL FLUID Ne 2047559211-11-2004GEREBRO SPINAL FLUID Ne 2047559112-11-2004CEREBRO SPINAL FLLTID Ne 2047558812-11-2004CEREBRO SPINAL FLUID Ne 2047558614-11-2004CEREBRO SPINAL FLUID Ne 2047557715-11-2004CEREBROSPINA.LFLUID Ne 2047562716-11-2004GEREBRO SPINAL FLUID Ne 2047569318-11-2004GEREBRO SPINAL FLUID Ne 2047574019-11-2004CEREBRO SPINAL FLUID Ne 2047568619-11-2004GEREBRO SPINAL FLUID Ne 2047568520-11-2004CEREBRO SPINAL FLUID Ne 2047572123-11-2004CEREBRO SPINAL FLUTD Ne 2047573624-11-2004CEREBRO SPINAL FLLTID Ne 2047579526-11-2004CEREBRO SPINAL FLUID Ne 204757962G-11-2004CEREBRO SPINAL FLUID Ne 2047580129-11-2004CEREBRO SPINAL FLUID Neg 2047584030-11-2004GEREBRO SPINAL FLUID Ne 2047586502-12-2004CEREBR.O SPINAL FLUID Ne 2047591103-12-2004CEREBRO SPINAL FLUID Neg 2047591703-12-2004CEREBRO SPINAL FLUID Ne 2047591403-12-2004CEREBRO SPINAL FLUID Ne 2047605403-12-2004CEREBRO SPINAL FLUID Neg 2047590504-12-2004CEREBRO SPINAL FLUID Ne 2047591006-12-2004CEREBRO SPINAL FLUID Neg 2047605310-12-2004CEREBRO SPINAL FLUID Ne 2047610313-12-2004CEREBRO SPINAL FLUID Ne 2047612216-12-2004CEREBRO SPINAL FLUID Ne 2047612716-12-2004CEREBRO SPINAL FLUID Ne 2047616617-12-2004CEREBRO SPINAL FLUID Neg 2047629322-12-2004GEREBRO SPINAL FLUID Ne 2047628625-12-2004CEREBRO SPINAL FLUID Ne 2047628527-12-2004CEREBRO SPINAL FLUID Ne 2047631228-12-2004CEREBRO SPINAL FLUID Ne 2057003131-12-2004CEREBRO SPINAL FLUID Ne 20570029O1-O1-2005CEREBRO SPINAL FLUID Neg ~

20570027O1-01-2005CEREBRO SPINAL FLUID Ne 2057010605-O1-2005CEREBRO SPINAL FLUID Neg 2057025705-O1-2005CEREBRO SPINAL FLUID Ne 2057025506-O1-2005CEREBRO SPINAL FLUID Ne 2057012807-O1-2005CEREBRO SPINAL FLUID Ne 2057025807-O1-2005CEREBRO SPINAL FLUID Ne 2057016808-O1-2005CEREBRO SPINAL FLUID Ne 2057025610-O1-2005CEREBRO SPINAL FLUID Ne 057016710-O1-2005CEREBRO SPINAL FLUID Ne 2057034612-O1-2005CEREBROSPINALFLUID Ne 2057035014-O1-2005GEREBRO SPINAL FLUID Ne 2057034315-O1-2005CEREBRO SPINAL FLUID Ne 2057044018-O1-2005CEREBRO SPINAL FLUID Ne 2057040318-O1-2005CEREBRO SPINAL FLUID Neg 2057048019-O1-2005CEREBRO SPINAL FLUID Ne 2057048320-O1-2005CEREBRO SPINAL FLUID Ne 2057048120-01-2005CEREBRO SPINAL FLUID Ne 2057047321-O1-2005CEREBRO SPINAL FLUID Ne 2057063126-01-2005CEREBRO SPINAL FLUID Ne 205705762G-01-2005CEREBRO SPINAL FLUID Ne 205706322S-O1-2005CEREBRO SPINAL FLUID Ne 2057062928-O1-2005CEREBRO SPINAL FLLTID Ne 2057062229-O1-2005CEREBRO SPINAL FLUID Ne 2057072931-O1-2005CEREBRO SPINAL FLUID Ne 20570730O1-02-2005CEREBRO SPINAL FLUID Neg 20570670O1-02-2005GEREBRO SPINAL FLUID Ne 2057072603-02-2005GEREBRO SPINAL FLUID Ne 2057082103-02-2005CEREBRO SPINAL FLUID Ne 2057082007-02-2005CEREBRO SPINAL FLUID Ne 2057084708-02-2005CEREBRO SPINAL FLUID Ne 2057080608-02-2005CEREBRO SPINAL FLUID Ne 2057085010-02-2005CEREBRO SPINAL FLUID Ne 2057088311-02-2005GEREBRO SPINAL FLUID Ne 2o57os8211-02-2005GEREBRO SPINAL FLUID Ne 2057088914-02-2005CEREBRO SPTNAL FLUID Neg 2057094414-02-2005CEREBRO SPINAL FLUID Ne 2057088114-02-2005CEREBRO SPINAL FLUID Ne 2057097617-02-2005CEREBRO SPINAL FLLTID Ne 2057097517-02-2005CEREBRO SPINAL FLUID Ne 2057102818-02-2005CEREBRO SPINAL FLUID Neg 2057108319-02-2005GEREBRO SPINAL FLUID Ne 2057117423-02-2005CEREBRO SPINAL FLUID Ne 2057107223-02-2005CEREBRO SPINAL FLUID Ne 2057107723-02-2005CEREBRO SPINAL FLUID Ne 2057111824-02-2005CEREBRO SPINAL FLUID Neg 2057116625-02-2005GEREBRO SPINAL FLUID Ne 2057123227-02-2005CEREBRO SPINAL FLUID Ne 20571226O1-03-2005CEREBRO SPINAL FLUID Ne 20571222O1-03-2005CEREBRO SPINAL FLUID Ne 2057125903-03-2005CEREBRO SPINAL FLUID Ne 2057131504-03-2005CEREBRO SPINAL FLUID Neg 2057136104-03-2005CEREBRO SPINAL FLUID Ne 2057131304-03-2005CEREBRO SPINAL FLUID Ne 2057131207-03-2005CEREBRO SPINAL FLUID Ne 2057136208-03-2005CEREBRO SPINAL FLUID Neg 2057138409-03-2005CEREBRO SPINAL FLUID Ne 2057143910-03-2005CEREBRO SPINAL FLUID Ne 2057145012-03-2005CEREBRO SPINAL FLUID Neg 2057151114-03-2005CEREBRO SPINAL FLUID Ne 205715411G-03-2005CEREBRO SPINAL FLUID Ne 205715101G-03-2005CEREBRO SPINAL FLUID Ne 2057159517-03-2005CEREBRO SPINAL FLUID Ne 2057159018-03-2005CEREBRO SPINAL FLUID Neg 2057158618-03-2005CEREBRO SPINAL FLUID Ne 2057159120-03-2005CEREBRO SPINAL FLL1ID Ne 2057162920-03-2005CEREBRO SPINAL FLUID Ne 2057165422-03-2005CEREBRO SPINAL FLUID Ne 2057165623-03-2005CEREBRO SPINAL FLUID Neg 2057165824-03-2005CEREBRO SPINAL FLUID Ne

Claims (44)

Claims

1. A method for reverse transcribing an RNA target molecule comprising incubating said RNA target molecule with a reverse transcriptase, preferably a moloney Murine Leukemia Virus reverse transcriptase or Superscript II or Superscript III or a functional part, derivative and/or analogue thereof in a solution comprising between 25 and 75 mM of a suitable salt, between 0.05 % and 0.2 % of a non-ionic detergent, between 60 and 240 µM per nucleotide or equivalent thereof 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 35 and 50 °C.

2. A method according to claim 1, wherein the solution is buffered at a pH between about 8 and 9.

3. A method according to claim 1 or claim 2, wherein the solution is buffered at a pH between about 8.1 and 8.5.

4. A method according to claim 3, wherein the solution is buffered at a pH of about 8.3.

5. A method according to any one of claims 1-4, wherein said solution further comprises random hexamer primers.

6. A method according to any one of claims 1-5, wherein said RNA
target molecule is derived from a faecal sample.

7. A method according to claim 6, wherein said RNA target molecule comprises an enterovirus and/or an influenza virus.

8. A method according to any one of claims 1-7, wherein said solution further comprises an internal control RNA molecule.

9. A method according to claim 8, wherein said internal control RNA
molecule comprises a sequence 5'-CCC TGA ATG CGG CTA ATC CTA ACC
ACG GAA CAG CTG GTC GCG AAC CAG TGA GCT 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'.

10. 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 said sample and with a first and a second primer, said first primer comprising a nucleotide sequence of a forward primer depicted in any one of tables G-13, or a functional equivalent thereof, and said second primer comprising a nucleotide sequence of the reverse primer depicted in the same table as said forward primer, or a functional equivalent thereof, and incubating said amplification solution to allow for amplification of said nucleic acid when present.

11. A method according to claim 10, wherein said virus comprises an enterovirus, adenovirus or influenza virus.

12. A method according to claim 10 or 11, wherein said first primer comprises a nucleotide sequence ID#1: 5'-CCC TGA ATG CGG CTA AT-3'; or a functional equivalent thereof and said second primer comprises a nucleotide sequence ID#2: 5'-ATT GTC ACC ATA AGC AGC C-3' or a functional equivalent thereof.

13. A method according to claim 10 or 11, wherein said first primer comprises the sequence 5'-CAGGACCTCCTCGGRCTTAYCTSACT-3' or a functional equivalent thereof and said second primer comprises the sequence 5'-GGAGCCACVGTGGGRTT-3' or a functional equivalent thereof.

14. A method according to claim 10 or 11, wherein said first primer comprises the sequence 5'-CTACAAGACCAATCCTGTCACYTCTG-3' or a functional equivalent thereof and said second primer comprises the sequence 5'-AAGCCTTCTACCTCTGCAGTCC-3' or a functional equivalent thereof.

15. A method according to any one of claims 10-12 or 14, wherein said sample comprises product of a method for reverse transcribing an RNA target molecule according to any one of claims 1-9.

16. A method according to any one of claims 10-15, further comprising analysing the presence or absence of said nucleic acid by means of a probe.

17. A method according to claim 16, wherein said probe comprises a nucleotide sequence of the virus-specific probe depicted in the same table as said forward primer and/or reverse primer, or a functional equivalent of said virus-specific probe.

13. A method according to claim 1G or 17, wherein the presence of 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.

19. A method according to claim 16 or 17, wherein the presence of nucleic acid derived from an adenovirus is analysed with a probe comprising a sequence 5'-CCGGGTCTGGTGCAGTTTGCCCGC-3'or a functional equivalent thereof.

20. A method according to claim 1G or 17, wherein the presence of nucleic acid derived from an influenza A virus is analysed with a probe comprising a sequence 5'-TTCACGCTCACCGTCTCCCAGTGAGC-3'or a functional equivalent thereof.

21. A method according to any one of claims 10-20, wherein said nucleic acid amplification solution further comprises an internal control nucleic acid molecule.

22. A method according to claim 21, wherein said internal control nucleic acid molecule comprises a nucleotide sequence of the internal control depicted in the same table as said forward primer and/or reverse primer, or a functional equivalent of said internal control.

23. A method according to claim 21 or 22, wherein said internal control comprises the sequence 5'-CCC TGA ATG CGG CTA ATC CTA ACC ACG GAA
CAG GCG GTG GCG AAC GAG TGA CTG GCT TGT CGT AAC GCG CAA
GTC TGT GCT TGA GAC GTG CGT GGT AAC CGT CCG TGT TTC GTG TTA
TTT TTA TCA TGG CTG CTT ATG CTTG ACA AT-3'.

24. A method according to any one of claims 21-23, further comprising analysing the presence or absence of said internal control nucleic acid by 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 IC-specific probe.

25. A method according to claim 24, wherein said IC-specific probe comprises the sequence 5'-CTT GAG ACG TGC GTG GTA ACC-3' , 5'-GATGTGTCCGCCGTGCTTCCCCTGG-3', or 5'-TTCACTGGGCCCCTACTCGCACTGAC-3', or a functional equivalent thereof.

26. A method according to any one of claims 10-25, wherein a product of said amplification is measured in real-time.

27. A method according to any one of claims 10-26, 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.

28. 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.

29. An aqueous solution according to claim 28, further comprising between 60 and 240 µM of at least one nucleotide or equivalents thereof and a reverse transcriptase, preferably a Moloney Murine Leukemia Virus reverse transcriptase or Superscript II or Superscript III or a functional part, derivative and/or analogue thereof.

30. A concentrate of an aqueous solution according to claim 28 or 29.

31. An isolated or recombinant oligonucleotide comprising a sequence as depicted in any one of tables 6-13.

32. An oligonucleotide according to claim 31 comprising a sequence 5'-CCC TGA ATG CGG CTA AT-3'; or 5'-ATT GTC ACC ATA AGC AGC C-3' ;
or 5'-GCG GAA CCG ACT ACT TTG GGT-3'; or 5'-CTT GAG ACG TGC GTG
GTA ACC-3' or 5'-CCC TGA ATG CGG CTA ATC CTA ACC ACG GAA CAG
GCG GTC GCG AAC CAG TGA CTG GCT TGT CGT AAC CTCG CAA GTC
TGT GCT TGA GAC GTG CCT GGT AAC CGT CCG TGT TTC CTG TTA TTT
TTA TCA TGG CTG CTT ATG GTG ACA AT-3.

33. A recombinant virus particle comprising a sequence as depicted in any one of Tables G-13 or a functional equivalent thereof, or the complement thereof.

34. A recombinant virus particle according to claim 33, comprising the 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 GAA GTC TGT GCT
TGA GAG GTG CGT GGT AAC CGT CCG TGT TTC CTG TTA TTT TTA TCA
TGG CTG CTT ATG GTG ACA AT-3' and/or ID#4 5'-CTT GAG ACG TGC GTG
GTA ACC-3' or the complement thereof.

35. A kit for detecting viral nucleic acid comprising at least one and preferably at least two, more preferably at least three oligonucleotides comprising a sequence as depicted in any one of tables G-13, or a functional equivalent of said sequence.

36. A kit according to claim 35 for detecting enteroviral RNA comprising at least one and preferably at least two, more preferably at least three oligonucleotides according to claim 32.

37. A kit according to claim 36, comprising an internal control nucleic acid, comprising a sequence according to claim 32, or a functional equivalent thereof.

38. A kit according to any one of claims 35-37, comprising an armoured internal control RNA.

39. A kit according to claim 35 for detecting adenoviral DNA comprising at least one and preferably at least two, more preferably at least three oligonucleotides comprising a sequence as depicted in table 11, or a functional equivalent thereof.

40. A kit according to claim 35 for detecting influenza A RNA
comprising at least one and preferably at least two, more preferably at least three oligonucleotides comprising a sequence as depicted in table 6, or a functional equivalent thereof.

41. A kit according to claim 39 or 40, further comprising an internal control nucleic acid, comprising a sequence as depicted in table 11 or table 6, or a functional equivalent thereof.

42. A kit according to any one of claims 35-41, further comprising an aqueous solution according to claim 28 or claim 29 and/or a concentrate actor ding to claim 30.

43. A kit according to any one of claims 35-42, further comprising Superscript II or Superscript III.

44. A primer pair comprising an oligonucleotide comprising a sequence of a forward primer as depicted in any one of Tables 6-13, or a functional equivalent thereof, and an oligonucleotide comprising a sequence of the reverse primer depicted in the same table as said forward primer, or a functional equivalent thereof.