The authors have declared that no competing interests exist.
Conceived and designed the experiments: MN DH AM CÖ JB. Performed the experiments: AM CÖ. Analyzed the data: AM CÖ MN. Contributed to the writing of the manuscript: AM CÖ MN.
Rotavirus infections are one of the most common reasons for hospitalizations due to gastrointestinal diseases. Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive. Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level. We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies. Twenty-two patient samples were analyzed and the assay showed high concordance with a PCR-based assay. In summary, we present a new assay for sensitive and variation tolerant detection of rotavirus.
Rotavirus infections are one of the most common infections seen in young children. By the age of five nearly 100% of all children, regardless of ethnicity or socio-economic status, have been infected at least once with rotavirus
Currently, enzyme immunoassays, latex agglutination assays, electron microscopy and reverse transcription polymerase chain reaction (RT-PCR) are commonly used for detection of rotavirus. However, all these tests have drawbacks. Latex agglutination assays and electron microscopy lack sensitivity
Here we present a new sensitive and rapid method for the detection of rotavirus, a dsRNA virus, from fecal samples. It is based on a modified version of a padlock probe-based assay targeting the ssRNA Crimean–Congo hemorrhagic fever virus
In this study, we successfully detected rotavirus, a highly variable dsRNA virus, with good sensitivity.
Rotavirus positive samples were previously detected with rotavirus antigen detection by enzyme-linked immunosorbent assay (ELISA) or 7-plex gastro VOCMA (multiplex PCR and liquid bead array), and the target region was further sequenced with a 3130 Genetic Analyzer (Applied Biosystem)
The fecal samples were collected and stored with the patients consent according to the Swedish Biobank Law (SFS 2002∶297) and were analyzed anonymously. All samples have been described in Öhrmalm
All Human Rotavirus A types that were available on NCBI blastn (National Library of Medicine) were used to design the padlock probes. Using the Consort program
(A) The sequence of the six padlock probes with the haplotyped degenerations and ligation site. (B) Alignment of 214 Human Rotaviruses. The variations in Rotavirus were mapped using BLASTn and ConSort. The length of the black bars represents the frequency of variation as an average percentage conservation at each nt position (y-axis).
Name | Sequence |
cDNA primer | 5′-Biotin-TGYARRTTCC AR+TTY+TCD+AT RTA-3′ |
Forward primer | 5′-GGCTTTW+AAA+ CGAA+GTC+TTC R-3′ |
PLP_A | 5′- |
PLP_B | 5′-GATAATTACW ATGAAGTGTA TGCAGCTCCT CAGTAGTGCG ACACATGACA TCAACCARTT TAAYCAAAT-3′ |
PLP_C | 5′-GATAGTWACY ATGAAGTGTA TGCAGCTCCT CAGTAGTGCG ACACATGACA TCAACCARTT TAATCAAAT-3′ |
PLP_D | 5′-GATARTTACY ATGAAGTGTA TGCAGCTCCT CAGTAGTGCG ACACATGACA TCAACCARTT TAATCAAAT-3′ |
PLP_6 | |
PLP_8 | |
Restriction oligo | |
Detection oligo | 5′- Cy3- |
Target_6 | 5′-Biotin-TTCATAGTAA GTATCATCTG ATTAAACTG-3′ |
Target_1 | 5′-Biotin-TTCATAGTAA TTATCATTTG GTTAAATTG-3′ |
A + sign before nt indicate the LNA residues. As implicated in PLP_A: the bold font indicated the Rotavirus targeting padlock probe arms, Italic font indicates the AluI restriction site, and the underlined sequence the sequence for detection probe hybridization.
A suspension of approximately 100 µl feces in 1 ml of TE- buffer was vortexed and centrifuged. 400 µl of supernatant was transferred into 2 ml of EasyMag lysisbuffer and total nucleic acid was extracted with the NucliSENS EasyMag extraction system (bio-Mérieux AB, Sweden) into an eluate of 110 µl, which was aliquoted and frozen at −70°C.
Two µl of nucleic acid extract was mixed with 2.2 µM of the biotinylated reverse primer in a total volume of 9 µl. The biotinylated RT-PCR reverse primer (nt position 448–426) Biotin-TGYARRTTCCAR+TTY+TCD+ATRTA (+ before nt indicates locked nucleic acid (LNA) residues) was ordered from Exiqon (Exiqon A/S, Denmark). The LNA residues were included to increase binding stability of the primer. To denature the dsRNA the samples were incubated for 5 min at 97°C followed by snap-cooling on ice for 2 min. The reverse transcription was performed in a total volume of 20 µl in 1x First strand buffer (Life Technologies), 2 mM dNTPs (Thermo Scientific), 5 mM DTT (Life Technologies), 40 U RNaseOUT (Life Technologies) and 200 U Superscript III (Life Technologies) at 50°C for 60 min. The reaction was inactivated by heating at 70°C for 15 min.
The presence of cDNA was analyzed by PCR using 200 nM of forward primer (nt position 1–23) GGCTTTW+AAA+CGAA+GTC+TTCR (+ before nt indicate the LNA residues) together with 200 nM of the biotinylated reverse primer, resulting in amplicons of 448 nt. Additionally, the PCR reaction contained 2 µl of first strand reaction, 2.5 mM MgCl2, 0.8 nM dNTP (Applied Biosystem), 1x buffer and 0.8 U of AmpliTaqGold (Life Technologies), in a total volume of 25 µl. The reactions were run at 94°C for 10 min, 50 cycles at 94°C for 30 s, 52°C for 30 s and 72°C for 30 s, ending with 72°C for 7 min. Products were analyzed with 1.5% EtBr agarose gel.
Prior to use, padlock probes were phosphorylated at the 5′ end. Padlock probes were incubated in a mixture of 10 U T4 Polynucleotide Kinase (Thermo Scientific), 50 mM Tris-HCl (pH 7.6 at 25°C), 10 mM MgCl2, 5 mM DTT, 0.1 mM spermidine and 1 mM ATP for 30 min at 37°C followed by 20 min at 65°C. Padlock probes were ligated onto their target by incubating a mixture of 2.5 µl of sample cDNA, 50 nM of each padlock probe, 0.2 µg/µl BSA (NEB), 5 U Ampligase (Epicentre) and 1x Ampligase reaction buffer (20 mM Tris-HCl (pH 8.3), 25 mM KCl, 10 mM MgCl2, 0.5 mM NAD, and 0.01% Triton X-100) at 50°C for 10 min. Prior to use, Dynabeads MyOne Streptavidin T1 beads (Life Technologies) were washed 3 times with washing buffer (10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% Tween 20, and 0.1 mM NaCl). Ten µl of 10 mg/ml Dynabeads were added to the ligated samples and incubated at room temperature for 5 min. All samples were washed once with washing buffer to remove unligated padlock probes. To replicate ligated circles, the washing buffer was discarded and replaced by a solution consisting of 0.2 µg/µl BSA, 125 µM dNTPs (Thermo Scientific), 100 mU/µl phi29 DNA polymerase (Thermo Scientific), and 1x phi29 DNA polymerase reaction buffer (33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 66 mM K-acetate, 0.1% Tween 20, 1 mM DTT). Polymerization occurred at 37°C for 20 min and was terminated at 65°C for 1 min. To monomerize the amplified RCPs, 5 µl of digestion mix (0.2 µg/µl BSA, 1x phi29 DNA polymerase reaction buffer, 0.6 U/µl AluI (NEB), and 600 nM restriction oligo) was added to the amplified RCPs and incubated at 37°C for 1 min followed by an inactivation step at 65°C for 1 min. Monomers were religated and a second round of RCA was carried out by adding 25 µl of 0.2 µg/µl BSA, 1.36 mM ATP, 28 mU/µl T4 DNA ligase (Thermo Scientific), 1x phi29 DNA polymerase reaction buffer, 100 µM dNTPs, and 120 mU/µl phi29 DNA polymerase. The mixture was incubated at 37°C for 20 min, followed by 65°C for 1 min. Undigested restriction oligos served as a ligation template and primer for the second RCA.
Cy3 coupled detection oligos were hybridized to the rolling circle products. Fifty µl of a labeling solution containing 40 mM EDTA, 40 mM Tris-HCl (pH 7.5), 0.2% Tween 20, 10 nM detection oligo, and 2 M NaCl was added and incubated at 70°C for 2 min, followed by 55°C for 15 min. Labeled rolling circle products were pumped through a micro-channel and visualized using a confocal microscope
The reaction scheme consists of several steps (
(1) RNA is extracted from patient samples and (2) cDNA is synthesized using biotinylated primers. (3) The DNA is denatured and (4) padlock probes are hybridized and ligated to their complementary target sequence. (5) This complex is captured onto magnetic beads to wash away unbound probes and (6) amplified by rolling circle amplification (RCA) for 20 min. (7) The rolling circle products (RCPs) are monomerized and (8) a second round of RCA is performed. (9) Fluorescently labeled oligonucleotides are hybridized to the RCPs for detection in a microfluidic setup using a confocal microscope.
Since rotavirus is a highly variable virus it is important to cover as many strain variants as possible in order to achieve an accurate diagnosis with a low false-negative rate which might be caused by non-matching padlock probes. Our approach uses a mix of padlock probes which all target the same region in the VP-6 gene. These padlock probes differ only in a few bases and are therefore able to compete with each other by binding to the same region. In cases of imperfect binding, especially at the end of the probe arms, ligation would be hampered and no rolling circle product would be generated from these targets. To investigate if this competition and inhibition actually occurs, thus, if an increase in the number of unique padlock probes results in a decrease in signal, we serially added the different batches of padlock probes to one amol of synthetic target (5′-Biotin-TTCATAGTAA GTATCATCTG ATTAAACTG-3′) and performed C2CA. Importantly, we did not observe a lower amount of RCPs with an increasing number of padlock probes (
Before testing the assay on patient samples the analytical sensitivity was estimated by preparing a 1∶10 dilution series from 106 to 103 copies of a short synthetic biotinylated target (5′ Biotin -TTCATAGTAATTATCATTTGGTTAAATTG-3′). This target is a not an ideal substitute for the real viral genome, but should be a relatively good substitute for the cDNA copy of the genome. Thus, any loss in cDNA synthesis efficiency is not taken into account with this target. C2CA was performed as described in materials and methods and 2.5 µl were analyzed in the microfluidic detection system. With the current setup the assay has a limit of detection of 1,000 molecules and a dynamic range of at least four magnitudes (
Short synthetic 5′ biotinylated DNA templates are serially diluted to assess the analytical sensitivity of our assay. The y-axis shows the number of rolling circle products (RCPs) and the x-axis the copy number of a synthetic biotinylated DNA target. The negative sample is a no template control. Error bars ±1 s.d.; n = 3.
One of the most important parameters for a diagnostic method is, next to specificity, clinical sensitivity. Ideally this should be 100%, but most molecular methods that are based on nucleic acid detection have a high risk of generating false negative results for highly variable viruses, such as rotavirus. Mismatches in probes or primers can result in a decreased or absent output signal. It is often difficult to combine several probes or primers to achieve complete coverage of possible strain variations. To investigate the tolerance for sequence variations using our padlock probe based approach, we tested samples from 20 patients diagnosed as rotavirus infected, and two patient samples diagnosed as rotavirus negative. The rotavirus positive samples contained samples from genogroup I (sample 2, 3, 4, 5, 17, 18 and 19) and genogroup II (sample 8, 9, and 10) of VP6. The other rotavirus positive samples could not be classified due to too short sequence information available. cDNA synthesis was confirmed by PCR followed by gel electrophoresis. All rotavirus samples positive by PCR were successfully identified (
cDNA was prepared from 22 patient samples collected at Uppsala University hospital. (A) PCR positive samples. (B) PCR negative samples. Plotted is the number of rolling circle product - (negative+3 SD) on the y-axis and patient samples on the x-axis. Error bars ±1 s.d.; n = 2.
To assess the robustness of the test in terms of analytical sensitivity and to determine the limit of quantification in clinical samples we performed a dilution series on sample number 1, 4, 6, 8, 10 and 14. Sample 1, 4 and 6 were even detectable when diluting 1∶1,000 whereas sample 8, 10 and 14 could only be diluted 100 times to be clearly above detection limit (
We hereby present a new rapid and sensitive assay for the detection of rotavirus in clinical samples with a total assay time of about 3.5 hours.
PCR assays are commonly used for nucleic acid detection, but they are limited in the degree of multiplexity and require two spatially separated recognition events, one for each primer. These properties render PCR assays less suitable for the detection of highly variable viruses. In contrast, padlock probes can be easily multiplexed by increasing the number of padlock probes and require only one recognition site. Even the emergence of new strain variants can be easily incorporated without tedious optimizations into the assay by simply designing a matching padlock probe and adding it to the existing pool of padlock probes.
Four out of 20 rotavirus positive samples did not yield any visible PCR product on the agarose gel. This could be due to failed cDNA synthesis, too low quality of the dsRNA or due to nonmatching primers. The specific primer for cDNA synthesis used in this assay could be replaced by random hexamers to make cDNA synthesis sequence independent, and thus create an assay even more tolerant to sequence variation. Sample 19 was detected by our padlock-based method, but not by PCR. This might be due to the fact that PCR requires two primers instead of one and is thereby less variation tolerant. The padlock-based assay is very versatile in forms of read-out. The microfluidic setup combined with a confocal microscope allows for a digital, quantitative read-out, but in many cases a yes or no answer is sufficient. Thus simpler read-outs, that are less quantitative, can be applied. One example of such a readout is the combination of padlock probes with the detection of horseradish peroxidase in a photometer
To summarize, we demonstrated for the first time the detection of a highly variable dsRNA virus using padlock probes.
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We thank Ronnie Eriksson for his input on padlock probe design.