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Abstract
Fast and accurate detection of viral RNA pathogens is important in apiculture. A polymerase chain reaction (PCR)-based detection method has been developed, which is simple, specific, and sensitive. In this study, we rapidly (in 1 min) synthesized cDNA from the RNA of deformed wing virus (DWV)-infected bees (Apis mellifera), and then, within 10 min, amplified the target cDNA by ultra-rapid qPCR. The PCR products were hybridized to a DNA-chip for confirmation of target gene specificity. The results of this study suggest that our method might be a useful tool for detecting DWV, as well as for the diagnosis of RNA virus-mediated diseases on-site.
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Fig. 1.
Location of primers for URRT-qPCR and probes for DNA-chip. The amplified target region is located at 9060–9282 bp on DWV RNA-dependent RNA polymerase. The size of the amplified target was 223 bp. The four DWV-specific probes represent the PCR product applied to the DNA-chip. DWV, deformed wing virus; PCR, polymerase chain reaction; URRT-qPCR, ultra-rapid reverse transcription quantitative polymerase chain reaction.
Fig. 2.
Comparison of the effectiveness of different reverse transcription reaction times. (A) Cts were compared to determine the efficiencies of cDNA synthesis with different RT reaction times in one-step or two-step RT-qPCR. (B) In one-step or two-step RT-qPCR, the Ct time was analyzed depending on the cDNA synthesis reaction time. Data are presented as mean ± strandard deviation values of three independent experiments. RT-PCR, reverse transcription-polymerase chain reaction; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
Fig. 3.
Optimal conditions for each step of DWV-specific URRT-qPCR. URRT-qPCR conditions were evaluated by examining Ct time values versus (A) annealing temperature, (B) annealing time, and (C) polymerization time. The annealing time was determined based on the stability of the detection system. Data are presented as mean ± strandard deviation values of three independent experiments. DWV, deformed wing virus; URRT-qPCR, ultra-rapid reverse transcription quantitative polymerase chain reaction.
Fig. 4.
Detection of DWV-specific target gene using URRT-qPCR. (A) DWV-specific URRT-qPCR was performed using recombinant DNA. The fluorescence signals of the amplified target gene were calculated to determine detection limits. Construction of a standard curve and (B) melting point analysis show that it differed the Tm valuese between specific and nonspecific amplicon. (C) The DWV-specific URRT-qPCR was performed with local samples and recombinant DNA, and it shows that Yeosu and Suwon sample have different values of Tm by melting point analysis. (D) For DWV-infected honeybee samples, we calculated the number of DWV molecules via regression analysis of the amplified target gene product. DWV, deformed wing virus; URRT-qPCR, ultra-rapid reverse transcription quantitative polymerase chain reaction.
Fig. 5.
Confirmation of amplified target gene presence on a DNA-chip. (A) Amplification of the target gene was confirmed on a DNA-chip by using four deformed wing virus-specific probes. The bar graph shows the SBR values, according to the number of recombinant DNA molecules. The SBR in each probe hybridized with deformed wing virus specific gene are shown. (B) Each sample was hybridized to each of the specific probes. The SBR values are shown by the graph. SBR, spot/ background ratio.
Table 1.
List of nucleotide sequences of the primers and probes used in this study
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