| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Molecular Diagnostics and Genetics |
Robert Koch-Institut,1 Projektgruppe, Neuartige Viren’, 2 Zentrum für Biologische Sicherheit 1, and 3 FG12 ‘Virale Infektionen’, Berlin, Germany.
aAddress correspondence to this author at: Robert Koch-Institut, Projektgruppe P11, Nordufer 20, 13353 Berlin, Germany. Fax 49-30-4547-2605; e-mail chmielewiczb{at}rki.de.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Methods: We developed a PCR-based assay that combines detection and genotyping of human AdVs, targeting a highly conserved region of the adenoviral genome coding for the DNA polymerase (AdV DPol PCR). We tested the diagnostic applicability of this PCR-based assay by analyzing 159 clinical specimens from children with respiratory disease and comparing the results with those obtained by nested PCR analysis.
Results: The PCR assay detected all currently known AdV serotypes, with a detection limit of 
10 genome equivalents per reaction for 49 of 51 serotypes. No cross-reactivity to human DNA or other DNA viruses was observed. In addition, genotyping of PCR-positive samples was achieved within minutes by fluorescence curve melting analysis in a LightCycler instrument using 6 pairs of hybridization probes, each specific for a single AdV species. Results for clinical specimens were in good concordance with those obtained by nested PCR.
Conclusion: The presented assay is a suitable tool for the detection and genotyping of human AdVs in clinical samples.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
AdVs are recognized as pathogens causing severe morbidity and mortality in immunosuppressed patients (4)(5)(6)(7). Up to 30% of these patients are found to be AdV positive after allogeneic hematopoietic stem cell transplantation, with a particularly high incidence of infection and viral disease in children (2)(4). Clinical signs of an AdV infection are rather nonspecific and variable and include tonsillopharyngitis, conjunctivitis, pneumonia, gastroenteritis, hepatitis, and hemorrhagic cystitis (2)(3)(4)(5)(8)(9)(10). Local infections generalize in some cases and then have a mortality rate of up to 60% (2)(3)(4)(5)(10)(11)(12).
Virus isolation in cell culture has been the gold standard for the diagnosis of AdV infection. This method allows the subsequent typing of the isolate by neutralization assays but is rather slow because some serotypes require up to 3 weeks to show a cytopathogenic effect (3)(4)(5)(9)(13). ELISA is commonly used for direct antigen detection in respiratory secretions and urine, but lacks sensitivity. An acute AdV infection can be detected by an increase in antibody titers in sequential blood samples, but this approach is not suitable for transplantation patients with an impaired immune response (2)(4)(5). PCR has become a valuable tool for the diagnosis of AdV infection because its speed, specificity, and sensitivity permit early onset of clinical treatment. Quantitative PCR assays allow monitoring of the kinetics of the viral infection to distinguish between acute and persistent infections and estimation of the success of antiviral therapy (4). Several qualitative, but only a few quantitative PCR assays for AdV detection have been reported, mainly targeting the hexon gene (3)(4)(9)(11)(12). Unfortunately, 2 of these quantitative assays fail to detect all serotypes, a third is rather complex to perform, and for the others the experimental data on the ability to detect all serotypes with comparable sensitivity are incomplete or totally absent. A theoretical estimation of generic PCR performance on the basis of sequence alignments is hindered by the lack of sequence information for a high number of AdV serotypes; therefore, for these serotypes the quantitative data are questionable. To overcome this diagnostic problem, we established a real-time PCR targeting a highly conserved region of the adenoviral DNA polymerase (DPol) gene, taking advantage of the minor groove binder (MGB) chemistry (5'-exonuclease principle). Subsequent fluorescence curve melting analysis (FCMA) of the obtained PCR products in a LightCycler instrument enabled identification of the adenoviral species.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
For verification of the serotype, a fragment of the hexon gene was amplified (14). The amplicon was sequenced directly, and the resulting sequence was compared with reference sequences in National Center for Biotechnology Information database. Serotypes lacking unequivocal matches with database entries were analyzed phylogenetically to verify correct classification into the assumed species.
DNA samples from herpes simplex virus-1 and -2, Epstein–Barr virus, cytomegalovirus, human herpesvirus (HHV)-8, and simian virus 40 were kindly provided by Bernhard Ehlers (Robert Koch-Institut, Berlin, Germany). Three clinical samples positive in PCR for transfusion-transmitted virus (TTV) DNA were obtained from Eckart Schreier, and the DNA for parvovirus B19 was from Oliver Donoso-Mantke (both from Robert Koch-Institut). DNA samples from HHV-6A, HHV-6B, and HHV-7 were provided by Aleksandar Radoni
(Charité, Berlin, Germany).
clinical samples
The clinical samples were part of the sample collection of the National Reference Center for Influenza (Robert Koch-Institut, Berlin, Germany). A total of 158 throat swabs and 1 throat wash were collected from 1999 to 2003 from children 0 to 9 years of age [0–4 years, n =145 (91.2%); 5–9 years, n = 14 (8.8%)] with fever and cough; 16 of the 159 samples had positive test results for AdV (viral culture plus antigen detection). Of the 159 samples, 105 were pooled into groups of 5 for DNA extraction. If the pool tested PCR positive, the 5 samples were extracted separately to identify the positive sample(s). All other samples, including the 16 samples positive for AdV, were extracted separately.
preparation and quantification of dna
DNA from viral cultures was extracted from 200 µL of culture supernatant by the DNA Blood Mini Kit (Qiagen) after addition of 2 µg of tRNA as a carrier, according to the manufacturer’s recommendations. The DNA was eluted in 200 µL of elution buffer.
Swabs taken from diseased children had been washed out in culture medium, which then was stored at –70 °C. DNA was extracted from 200 µL of the culture medium after addition of 2 µg of tRNA and eluted in two 50-µL volumes of elution buffer. For DNA extraction from sample pools, 5 samples (40 µL each) were mixed, and DNA was extracted as described above.
Plasmid DNA was extracted from 1 mL of bacterial culture by use of the NucleoSpin Kit (Macherey & Nagel), including the additional washing step (AW buffer), as recommended by the manufacturer.
For sequencing, PCR products were quantified in agarose gels; for serial dilutions, plasmid DNA and PCR products were quantified fluorometrically (Turner Designs).
The human DNA for determining assay specificity was purchased (human placenta DNA; Sigma Aldrich).
pcr
All oligonucleotides used are listed in Table 1
of the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol51/issue8/. The MGB probes were synthesized by Applied Biosystems; all other oligonucleotides were synthesized by TIB MOLBIOL.
|
Conventional PCR.
PCR reactions were set up in a total volume of 25 µL containing 1x PCR buffer, 2 mM MgCl2, 1 mM deoxynucleotide triphosphates with dUTP, 500 nM each primer, 0.5 U of Platinum Taq Polymerase (Invitrogen), and 1–10 µL of template DNA. Amplification was carried out in a GeneAmp 2400 or 9600 Instrument (Applied Biosystems) for a total of 45 cycles. After an initial denaturation step at 95 °C for 5 min, each cycle consisted of denaturation at 95 °C for 30 s, followed by annealing at a primer-specific temperature for 30 s and primer extension at 72 °C for 30 s. The PCR product was analyzed on a 2% agarose gel and visualized with ethidium bromide under ultraviolet light.
Nested PCR.
Nested PCR on clinical samples was performed with primers described by Allard et al. (14) with slight modifications (P-029 and P-030). Sequences of both first-round primers and the second-round antisense primer were modified according to the available sequence data. Reaction conditions were as described for conventional PCR with 1 µL of the first-round PCR product being transferred to the second-round PCR. In both rounds, 40 cycles were performed.
Quantitative PCR.
Quantitative real-time PCR was carried out on an ABI Prism 7700 or 7900HT sequence detection system (Applied Biosystems) in a total reaction volume of 25 µL. The reaction contained 1x PCR buffer, 6 mM MgCl2, 1 mM deoxynucleotide triphosphates with dUTP, 1 µM ROX, 0.5 U of Platinum Taq Polymerase (Invitrogen), 100 nM each primer, and both MGB probes at a concentration of 50 nM. After 5 min at 95 °C for Taq DNA polymerase activation and DNA denaturation, a total of 40 cycles consisting of denaturation at 95 °C for 15 s, annealing at 60 °C for 20 s, and primer extension at 72 °C for 30 s were performed. For quantification we used serial dilutions of one of the plasmids (106 to 101 genome equivalents). After the run, data were analyzed by use of Sequence Detector 1.6.3 or SDS 2.2 software, respectively.
fcma
For AdV-positive samples, FCMA was performed in a LightCycler instrument (Roche). PCR product (1–5 µL) was added to the melting mixture consisting of Platinum PCR buffer (final concentration: 1x), 5 mM MgCl2, and 150 nM each probe (listed in Table 1
of the online Data Supplement) to a final volume of 10 µL. The samples were denatured at 85 °C for 30 s, cooled to 35 °C at maximum ramping rate (20 °C/s), and reheated to 85 °C at a ramping rate of 0.2 °C/s, during which the fluorescence data were acquired. Finally, the samples were cooled to 35 °C. The data were analyzed by use of LightCycler software (Ver. 3.5).
cloning of pcr products
Fresh PCR products (P-025/026 or P-038) were cloned by use of the TOPO TA Cloning Kit (Invitrogen) according to the manufacturer’s instructions.
sequence determinations
After purification (PCR Purification Kit; Qiagen), amplicons were directly sequenced by dye terminator chemistry (ABI-Prism Big Dye Terminators v3.1 Cycle Sequencing Kit; Applied Biosystems) in an ABI Prism 3100 genetic analyzer (Applied Biosystems). Amplicons for which yield or purity was not sufficient for direct sequencing were cloned by use of the TOPO TA Cloning Kit (Invitrogen). The resulting plasmids were sequenced by use of primers M13 and T7 supplied by Invitrogen. The obtained sequences were analyzed, assembled, and aligned by use of the DNA STAR software package. All new sequences presented in this study were determined by sequencing of at least 2 independent PCR products in both directions, i.e., each nucleotide was determined at least 4 times.
accession numbers
Sequence data from this report have been deposited with the GenBank/EMBL/DDBJ data libraries. All accession numbers are listed in Table 2
of the online Data Supplement. The amino acid sequences of these residues were deduced.
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
of the online Data Supplement, the designated probe binding site is highly conserved. The variable nucleotide at position 10 in the 21-nucleotide stretch is either adenine or guanine in all serotypes, and only 1 serotype (AdV 18) has an additional variation at position 6. We therefore chose this 21-nucleotide region as the probe binding site by applying the MGB chemistry. Because of the variable nucleotide in the middle of the probe target sequence, we used 2 probes that differed in the variable position but were identical elsewhere.
|
In proximity to the probe target, the sequences were too variable to be detected by a single primer pair. We therefore designed 5 primer pairs (P-033, P-034, P-035, P-039, and P-040) to ensure amplification of all 51 serotypes. These primers give an amplicon with a length of 115 bp. When a mixture of these 5 primer pairs and the 2 probes at an annealing temperature of 60 °C (3–6 °C below the melting temperature of the primers) was used, all 51 serotypes could be amplified efficiently.
evaluation of the pcr assay
A fragment of the DPol gene of 6 serotypes representing the 6 species (AdV 2, 3, 4, 9, 12, and 40) was amplified by use of the primers P-025/P-026 and cloned into the TA cloning vector. These primers are located upstream and downstream of the PCR target region so that the plasmids contain viral sequences that are not affected by potential primer artifacts in the assay’s binding sites. Examination of a 10-fold dilution series of these plasmids (106–101 genome equivalents in 1 ng/µL 
DNA) revealed a linear detection range for all 6 serotypes with comparable detection limits (Fig. 1
, top panel). All reactions with dilutions containing 102 genome equivalents gave positive amplification, and the dilutions containing 101 genome equivalents were successfully amplified in 64 (88.9%) of 72 reactions (12 reactions per serotype, failure of detection was distributed over all 6 serotypes). In all runs, the correlation (r2) exceeded 0.98 even if it was calculated on the basis of all 6 plasmid dilution series (Fig. 1
, bottom panel).
To further analyze assay performance, we examined dilutions of all 6 plasmids in quadruplicate in a single run (for intraassay imprecision) and in duplicate in 3 independent runs (for interassay imprecision). The intra- and interassay imprecision was very low, even for low copy numbers (summarized in Table 1
). We also calculated interplasmid variability to evaluate the differences in detection limits for the 6 serotypes. As shown in Table 1
, the variability of threshold cycle values between the 6 different targets also was very low, even for low copy numbers. Using the data collected during these runs, we calculated that PCR efficiency was 99.8% [mean (SD) slope, –3.32 (0.074) with all 6 serotypes included].
To assess the sensitivity of the PCR assay regarding the remaining 45 serotypes, we used the amplicons and plasmids produced for sequence determination. We first measured the DNA concentration fluorometrically to determine the copy number of PCR products as accurately as possible. We then examined serial dilutions with 106 to 101 copies per reaction, and 43 of 45 (in total, 49 of 51 serotypes) were detected with an almost identical sensitivity. For serotype 16, which has a mismatch at the second position of the 3' end of the reverse primer, the lowest detectable concentration was 10-fold higher. The same result was observed for serotype 18, which has a mismatch in the probe sequence, that also caused a decrease in fluorescence intensity (data not shown).
To elucidate the influence of human DNA on the detection of AdV in clinical samples, we added 100 ng of human placental DNA per reaction to the plasmid dilutions. This procedure did not cause a markedly changed threshold cycle for any of the 6 plasmid dilutions. The human DNA itself was not amplified, as seen in reactions without AdV plasmids.
We evaluated the specificity of the assay by testing DNA from several DNA viruses. The PCR assay did not cross-react with herpes simplex virus types 1 and 2, Epstein–Barr virus, cytomegalovirus, HHV-6 A and B, HHV-7, HHV-8, simian virus 40, parvovirus B19, camelpox, cowpox, modified vaccinia virus Ankara (MVA), or 3 clinical samples that were PCR-positive for transfusion-transmitted virus (data not shown).
fcma for identification of the ADV species
To enable further characterization of the amplified viral sequences, we used FCMA in a LightCycler instrument to identify the AdV species. A set of 6 hybridization probes, all located within the amplicon, was designed. Each of these probe pairs was specific in sequence for 1 of the 6 species but had mismatches to the others. As a consequence, PCR products with sequences that were perfectly complementary to the sequences of the hybridization probes showed the highest possible melting temperatures, whereas PCR products with differing sequences (representing 1 of the 5 other species) had a lower melting temperature or no melting peak at all (Fig. 2
and Table 2
).
|
To perform FCMA, the obtained PCR product was transferred to a melting mixture (see Materials and Methods) in a LightCycler capillary. For every PCR product, 6 reactions were run, each containing 1 of the 6 probe pairs. The run in the LightCycler instrument took <10 min and gave a melting pattern composed of the melting temperatures that were obtained with the 6 different probe pairs. This melting pattern was characteristic for the amplified viral sequence, so that the AdV species could be easily deduced. The melting temperatures of all serotypes are listed in Table 2
.
clinical samples
To evaluate the performance of the assay for clinical samples, we examined 159 samples from children with respiratory diseases. The samples were reexamined by a generic nested PCR, according the method described by Allard et al. (14) with slight primer modifications. All results are summarized in Table 3
. In total, 37 of the 159 samples (23.3%) were positive, including all 16 samples previously testing as positive. Of these 37 samples, 35 were from children 0–4 years of age (35 of 145; 20.7% of this age group) and 2 were from children 5–9 years of age (2 of 14; 14.3% of this age group).
|
Our assay detected adenoviral DNA in 32 samples (see Table 3
); 24 of these samples also gave signals in the first round of the nested PCR, whereas 7 samples were positive only after second-round PCR. One sample was positive with our assay but negative with the nested PCR. All 5 samples that were negative with our assay but positive with the nested approach gave signals only after second-round PCR; their viral load was therefore considered to be very low.
The FCMA results for PCR products after AdV DPol PCR were in complete concordance with the results obtained by sequencing of the nested PCR products. The majority of the detected viruses belonged to species C [23 of 37 amplicons (62.2%)] or B1 [12 of 37 amplicons (32.4%)]. One amplicon was found to be derived from a species F and 1 from a species D virus (2.7% each).
Statistical comparison of the 2 methods of AdV DPol PCR and hexon nested PCR gave a specificity of 96.1%, sensitivity of 96.9%, positive predictive value of 86.1%, and negative predictive value of 99.2%.
To elucidate the extent of sequence conservation in circulating virus strains, we sequenced the AdV DPol PCR target regions of 22 viruses detected in the respiratory samples with primers P-038 (data not shown). All species C viruses (12 samples) were completely identical to our prototype viruses in the PCR target region. In contrast, 2 nucleotide changes were observed in the sequences of species B1 viruses (10 samples), both located within primer binding sites. One change, in the sense orientation, affected 1 nucleotide (position 9 of the 3' end) to which the primer already had a mismatch compared with our prototype viruses (G/C observed in 1 of 10 clinical samples instead of G/A in the prototype and in 9 of 10 clinical samples). In contrast, the second mismatch, in the antisense orientation (position 11 of the 3' end), was observed in 9 of 10 samples. As expected from the FCMA results, none of the amplified viruses had any sequence variabilities within the hybridization probe binding sites.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
On the basis of the published sequence information, we examined various genes of the viral genome as possible target sequences for a quantitative PCR system. We focused on genes involved in DNA replication because these genes are known to be highly conserved (17)(18)(19). In alignments of published nucleotide sequences, only 1 region seemed suitable as a target for a generic probe. This stretch of 21 nucleotides is located in the C-terminal part of the adenoviral DPol gene between a suggested DNA binding domain (4) and a region that is highly conserved in family B 
-like DNA polymerases (conserved region 1/Pol 1) (18)(19).
Sequencing of the corresponding genome region (300 bp) of all 51 serotypes revealed a comparably high degree of sequence identity. As observed for other parts of the genome (2)(3)(6)(7)(20), the sequences were nearly identical within each species but varied with respect to the other species. On the basis of the generated sequence information, we chose 5 primer pairs that flanked the selected probe target sequence. They were designed so that no serotype differed from 1 of the 5 primers in more than 3 nucleotides. Considering these mismatches, we chose an annealing temperature below the melting temperature of the primers (60 °C). At this temperature, there was efficient amplification of the viruses tested in this study, and it was also possible to detect circulating virus strains with additional sequence variations.
The selected probe binding site was extremely conserved among all 51 serotypes. Apart from the variable A/G position in the middle of the probe, only AdV 18 has a second variation. Because this region lies adjacent to putatively functional domains of the DNA polymerase and because the exchange between adenine and guanine at the variable position does not produce an amino acid change, it is tempting to speculate that this part of the open reading frame codes for a protein domain that is important for the catalytic activity of the protein. Thus, it is unlikely that viruses circulating in the population have sequence variations at this particular site that might impair their detection.
To overcome the problem of the conserved sequence stretch being too short and AT rich for the use of a standard DNA-based 5'-exonuclease probe, MGB technology was applied. The MGB molecule is linked to the 3' end of the probe and stabilizes its binding to the target sequence by hydrophobic interactions. The resulting increase in the melting temperature of the probe allows for the design of shorter probes, thereby opening new opportunities for the detection of highly variable sequences (21). Because the short sequence of the MGB oligonucleotide and its strong binding sensitize the probe to mismatches, the use of only 1 probe was insufficient for detection of those viruses having the diverging sequence. We therefore introduced a second, identically labeled probe that covered the differing sequences and dramatically improved their detection.
Using the mixture of 5 primer pairs and 2 probes, we evaluated the assay in regard to several diagnostically relevant characteristics. It allowed reproducible detection of dilution series of plasmids or PCR products of 49 of 51 serotypes within a linear detection range from 106 to 101 genome equivalents per reaction and showed only negligible differences in the amplification of different species or serotypes. Only 2 serotypes (AdV 16 and AdV 18) had lowest detectable concentrations that were 10-fold higher because of sequence variations at relevant positions. Furthermore, the assay is highly specific for AdVs because no other DNA viruses or human genomic DNA are amplified.
In consideration of the variability of the underlying sequence data and the complexity of the assay design, the good performance characteristics described here might greatly depend on the reagents used for amplification. Taq polymerases have been reported to exhibit different efficiencies in real-time PCR formats (22), a characteristic that might lead to impairment of AdV detection if our AdV DPol assay is used with a modified protocol. Nevertheless, this assay is a suitable tool for generic AdV detection and allows the identification of positive samples within a few hours. However, it only reveals the presence of AdV and does not allow further differentiation of the AdV species or serotypes, which can be of interest for the clinician. Because the AdV serotypes within a species are similar in tropism, pathogenic potential, and tendency to cause persistent infection (14)(23), it is generally accepted that identification of the serotype is dispensable because the same information is given by identification of the AdV species (4)(23)(24). Thus, to allow for further characterization of the amplified virus, we developed a method for genotyping on the species level that is based on the DPol PCR and takes advantage of the DNA sequence characteristics of this virus family.
Today, in many laboratories, genotyping by sequencing or restriction enzyme analysis of PCR products has replaced serotyping of a virus isolate by neutralization assays (9)(14)(20)(23)(25). These molecular methods are much faster, but they still take a few days until a diagnosis is confirmed. Because time is critical, particularly for the diagnosis of viral infections in immunosuppressed patients (4), we established a protocol for rapid genotyping of the amplified virus by use of FCMA. This methodology, usually applied for single-nucleotide polymorphism analysis after amplification in a LightCycler, was adapted to differentiate the AdV DPol PCR amplicons with regard to the AdV species, based on the fact that the sequences of serotypes that are grouped into a species are highly homologous but differ from those of the other species. Consequently, a hybridization probe pair that is specific for one species has mismatches to the others, giving lower melting temperatures. Generally, these melting temperatures are characteristic for the target sequence/probe-pair combination, making them highly reproducible. The parallel analysis of an amplicon with the different probe pairs will therefore give a characteristic melting pattern of 1 perfect match and 5 nonperfect matches, from which the species can be easily deduced. In single cases even the serotype can be identified. Furthermore, because they differ in melting temperature, the 2 subspecies B1 and B2 can be differentiated with the probes for species F.
The examination of clinical samples underscored the good performance of our assay. From 159 samples examined, we obtained a total of 37 amplicons, which were derived almost exclusively from species C and B1 viruses, with the exception of 1 species F and 1 species D virus. Species C and B1 viruses are a common cause of respiratory disease (26) and therefore were expected to be frequently observed. However, species F and D viruses usually are isolated from patients with gastrointestinal disease or conjunctivitis, respectively, and only infrequently cause respiratory symptoms (27)(28)(29). In total, the results of our assay were in good concordance with those obtained with the nested PCR. Only 5 samples were identified in which a product was obtained by nested PCR but our assay did not detect AdV; however, these samples were positive only in second-round PCR, indicating that the copy number of AdV in these samples was very low. In addition, for the 31 samples that were positive with both assays, sequencing of the nested PCR amplicons confirmed the results obtained by FCMA.
Because we determined the serotype melting patterns by analyzing only one prototype of each AdV serotype, we cannot exclude the possibility that AdV strains exist that show sequence variations in the hybridization probe binding region and therefore give altered melting results. However, we consider this option to be rather unlikely because a particularly conserved region of a highly conserved gene in a DNA virus served as the PCR target. To support this hypothesis, we sequenced the viruses in a selection of 22 respiratory samples. The result confirmed the high extent of homology in this genome region because all viruses were found to be completely (species C) or nearly (species B1) identical to our prototype viruses. Although 2 nucleotide changes were observed for species B1 viruses in the primer binding regions, we do not consider these to significantly impair the detection of viruses because they are not located directly at the 3' end of the primer sequences and, furthermore, the criterion of a maximum of 3 mismatches to any primer is still fulfilled. This criterion was defined for the primer design and was sufficient to ensure the amplification of all serotypes when the described reaction conditions were applied. Furthermore, neither the MGB probe binding site nor the FCMA hybridization probe binding sites showed any sequence variabilities. In total, no limitation of detection and genotyping would be expected from the sequence data of these circulating viral strains. However, our survey of circulating AdVs was not exhaustive, and viruses with diverging sequences, although rare, may be present. Although in that case precise identification of the species would not be possible, nevertheless, some species could clearly be excluded. For the clinician, this exclusion process might be a productive course of analysis until other results, molecular or cell culture based, are acquired that further characterize the virus.
The presented assay combining generic real-time PCR and FCMA is a tool for the rapid, specific, and sensitive detection and genotyping of AdVs in clinical samples. In our assay, the PCR step reliably reveals the presence of adenoviral DNA within 1.5 to 2 h, depending on the instrument used. In addition to the speed of detection, determination of the viral load is valuable because it may have predictive value for disseminated AdV disease (3)(4)(7)(9)(12)(13). We undertook an extensive validation effort, not only to ensure amplification of all targeted viral serotypes but also to demonstrate accurate quantitative results across all serotypes. For further characterization, comparably time-consuming molecular methods such as sequencing are replaced by FCMA, by which the AdV species can be identified within minutes. A clinical sample can be analyzed within only 3 h, a total time that represents the most rapid experimental protocol described to date for the detection and genotyping of human AdVs and allows the timely onset of antiviral therapy, which has been associated with reduced risk of treatment failure (4)(8). In addition, other measures such as isolation of infected individuals, e.g., during outbreaks of respiratory AdV disease in military facilities, can be accomplished earlier and additional infections thus prevented. Taken together, the features of this assay can contribute to substantial improvement of the surveillance and clinical outcomes of patients at risk for AdV disease.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
