The Senzhang strain (GenBank accession no. AM600965, Far-Eastern subtype) preserved in our lab was used as the reference standard. Vero E6 (ATCC CRL-1586) cells cultivated in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum, 1% glutamine, 1% penicillin, and 1% streptomycin were infected with the TBE virus (multiplicity of infection = 1). After 3-5 days, the cell culture supernatant was harvested. Viral titers [plaque-forming units (pfus)] were determined using a plaque assay, as described by Cao et al..
In total, 203 full-length sequences of TBE were downloaded from GenBank and aligned using ClustalX ver. 2.0. The most conservative regions were identified manually for designing primers and probes. Primer Express 3.0 (Applied Biosystems, Foster City, CA, USA) was used to evaluate the physical properties of the primers and probes. Ultimately, the primers and probes were designed based on the NS5 region of the Far-Eastern subtype sequence (Table 1). All primers and probes were synthesized and purified by high-performance liquid chromatography (HPLC) by Sangon Biotech (Shanghai, China).
Primers/Probes Sequence (5′-3′) Genomic Region Genomic Position Product Size (bp) TBE-F CCTTTGGACAGCAGCGAGTGTTCAARGAGAA NS5 8708-8739 TBE-R CTATGAAYTCCTCTCTGCTGCACATTCGTGG NS5 8836-8866 159 TBE-Probe* AGGCTCAGGAGCCTCAGCCTGGCACAARGG[FAM-dT](THF)A [BHQ-dT] CATGAGAGCAGTGAATGA (C3-spacer) NS5 8750-8801 Note. *Probe modifications: FAM, 6-carboxyfluorescein; THF, tetrahydrofuran; BHQ, black hole quencher; C3-spacer, 3′ phosphate blocker.
Table 1. Sequences of Primers and Probes for RT-RAA Assays for TBEV
Targeted NS5 gene fragments were cloned into linearized pGEM-T Easy Vectors (Promega, Madison, WI, USA) with T4 DNA ligase. Linearized plasmid DNA template was recovered, purified, and enriched with MagicPure size-selection DNA beads according to the manufacturer's specifications. To recover RNA transcripts, the DNA templates were transcribed with T7 polymerase using the T7 High Efficiency Transcription Kit (TransGen Biotech, Beijing, China) and treated with DNase I to remove any residual DNA. Subsequently, the RNA copy number was calculated according to a previously described method[25, 26]. The RNA transcripts were used to evaluate the sensitivity of the RT-RAA.
All viral RNA was extracted from cell cultures and tick specimens using the QIAamp Viral RNA Mini Kit (QIAGEN, Dusseldorf, Germany) according to the manufacturer's specifications. The RT-RAA reaction was performed with an RT exo kit (Jiangsu Qitian Bio-technology, China), which mixed all the enzymes (SSB, 800 ng/μL; UvsX, 120 ng/μL; DNA polymerase, 30 ng/μL) for the reverse transcription and DNA amplification in lyophilized form in one tube. The reaction mixture contained 1 μL of RNA template, 25 μL of rehydration buffer, 16.7 μL of diethyl pyrocarbonate (DEPC) H2O, 2.1 μL of each primer (10 μmol/L), and 0.6 μL of probe. Finally, 47.5 μL of master mix/template solution was transferred to each lyophilized enzyme mix. Then, 2.5 μL of 280 mmol/L magnesium acetate was pipetted into each tube lid. The tube lids were closed carefully, vortexed briefly, and centrifuged simultaneously to trigger the RT-RAA reaction. The tubes were then transferred to an RAA fluorescence detection device QT-F1620 with FAM and HEX dual-channel detection equipment (Jiangsu Qitian Bio-technology, China) at 39 ℃ for 30 min. A negative control (nuclease-free water) was included in each run.
Dilution series with standard RNA transcripts ranging from 106 to 100 copies RNA/reaction and TBEV titers between 103 and 100 pfu per reaction were prepared and used in the real-time fluorescence detection assay to evaluate the sensitivity of the RT-RAA. Eight replicates were performed for each dilution. The limit of detection was calculated by probit analysis at 95% probability.
To test the specificity of this assay, cross- reactivity was examined with Japanese encephalitis virus, Culex flavivirus, West Nile virus, Zika virus, Yellow fever virus, Dengue virus (serotypes 2), Tahyna virus, Getah virus, Sindbis virus, and Kadipiro virus which preserved in our lab. The accuracy of the RT-RAA assay were performed with previous preserved tick samples and Taqman RT-PCR method[13, 27].
Design of Primers and Probes
Production of a Standard Control
RNA Extraction and RT-RAA
Specificity and Sensitivity of RT-RAA
Only the TBEV RNA produced amplification signals, whereas none of the other arboviruses and no-template controls produced a positive amplification signal (Figure 1).
The sensitivity of the TBEV titers was determined by serial 10-fold dilution with concentrations ranging from 1 × 103 to 1 × 100 pfu per reaction (Figure 2). The limit of detection of the TBEV titers at 95% probability was 3 pfu per reaction (probit analysis, P ≤ 0.05) (Table 2). Similarly, the sensitivity for in vitro-transcribed RNAs was observed with serially diluted concentrations ranging from 1 × 106 to 1 × 100 copies per reaction (Figure 1). The limit of detection of the in vitro-transcribed RNAs at 95% probability was 20 copies per reaction (probit analysis, P ≤ 0.05) (Table 2).
Figure 2. Sensitivity of the RT-RAA assay for TBEV. A panel of serial 10-fold dilutions of (A) transcript RNA from 106 to 100 copies per reaction and (B) TBEV titers from 103 to 100 pfu per reaction was used to determine the sensitivity of the RT-RAA assay. NC, negative control.
Concentration No. of Positive Samples /No. of Samples Tested by the RT-RAA Assays for Detection of TBEV TBEV titers* RNA transcripts** 103 8/8 8/8 102 8/8 8/8 101 8/8 6/8 100 6/8 0/8 Note. Each dilution was tested in a total of 8 replicates; Concentration unit: *pfu per reaction; **copies per reaction.
Table 2. Assay Data Used for Probit Analysis to Calculate the Detection Limit of RT-RAA for TBEV
TaqMan RT-qPCR was performed to verify the accuracy of our assay. Ten batches of ticks collected in the Changbai Mountains in 2011 were used; three of them were identified as positive for TBEV, which was consistent with the results of the quantitative real-time PCR assay. The results indicated that the sensitivity and accuracy of the RT-RAA assay developed in this study were the same as those of TaqMan RT-qPCR (Table 3).
Year Location Strain No. of Ticks TaqMan RT-PCR RT-RAA 2011 Changbai JLP04 10 Neg Neg 2011 Changbai JLP08 10 Pos Pos 2011 Changbai JLP17 10 Neg Neg 2011 Changbai JLP22 10 Neg Neg 2011 Changbai JLP35 10 Pos Pos 2011 Changbai JLP36 10 Neg Neg 2011 Changbai JLP40 10 Pos Pos 2011 Changbai JLP51 10 Neg Neg 2011 Changbai JLP52 10 Neg Neg 2011 Changbai JIP53 10 Neg Neg Note. Pos: positive; Neg: negative.
Table 3. Comparison of TBEV Detection in Ticks Examined RT-RAA and TaqMan RT-PCR