DBPIA-NURIMEDIA

Original Article Journal of Apiculture 31(2) : 121~131 (2016) The Most Rapid Detection Method against Korean Sacbrood Virus using Ultra-Rapid Reverse-Transcription Real-Time PCR (URRTRT-PCR) Sang-Hyun Min, Jee-Hee Wang, Su-Jin Lim, Chil-Woo Lee* and Byoung-Su Yoon* Department of Life Science, College of Natural Science, Kyonggi University, Suwon, Republic of Korea (Received 26 April 2016; Revised 23 June 2016; Accepted 28 June 2016) Abstract New strain of Sacbrood virus as pathogen of honeybee, named korean Sacbrood virus (ksbv) was firstly recognized in Korea on 2010. During the time 2010-2015 near 90% populations of Apis cerana in Korea were heavy damaged by infection of ksbv. It is not clear yet, how to prevent it or how to control it and why infect only to A. cerana. These are why this development of rapid and easy detection method is important. In this study, the fastest and easy detection method against ksbv was designed. To optimize Ultra-Rapid Real-Time PCR (URRT-PCR) and cdna generation the ksbv-detection time from ksbv-infected larvae could be minimized. Using optimized ksbvspecific ultra-rapid reverse transcription real-time PCR (ksbv-urrtrt-pcr), minimum detection time for ksbv-infection was completed inner 6 minutes 12 seconds from ksbv-infected larvae quantitatively. Alternatively 2.5 10 1 copies of cdna from ksbv-genome could be detected using developed method. ksbv-urrtrt PCR might be helpful to detect ksbv quantitatively in laboratory and also in apiary field. Key words: Korean sacbrood virus, URRT-PCR, Reverse transcription, Rapid detection, Honeybee (Sacbrood virus; SBV) 1913, 1964 (Bailey, 1964). iflavirus RNA(positive strand RNA) picornavirus (Ghosh et al., 1999). SBV, (Sac), (Bailey et al., 1975). *Co-corresponding author. E-mail: bsyoon@kyonggi.ac.kr; chilwoo.lee@gmail.com 121

122 (Apis mellifera), (Apis cerana), (Chinese sacbrood virus; csbv) (Ma et al., 2011), Apis cerana (Korean sacbrood virus; ksbv) (Han et al., 2011).,,,.. PCR, PCR (Real-time PCR; RT-PCR),., chip PCR PCR (thermal cycler), GenSpector TMC- 1000(Samsung, Korea) PCR. - (glass-silicone based micro-chip) 6µl PCR,, PCR (Cho et al., 2006). PCR(Ultrarapid real-time PCR; URRT-PCR), PCR, 100µl, PCR cycle,, two-step URRT-PCR, (Escherichia coli) stx2 7 8, Vibrio parahaemolyticus tdh 7 10 PCR (Kim et al., 2010a; Kang et al., 2010b)., PCR,, (step), PCR (cycles) PCR,, DNA (Extreme thermophile DNA polymerase)., RNA,, RNA cdna.,, RNA, reverse transcription, PCR, PCR (URRTRT-PCR; Ultra-Rapid Reverse- Transcription Real-Time PCR). ksbv (Apis cerana) (larvae),. ksbv, ksbv Genbank accession Number HQ322114. 1.5ml 2 homogenizer. intron R&A-Blue

123, RNA,, 13,000 rpm, 10, 4 C, RNA. RNA (spectrophotometer),, 70 C. cdna RNA 1µg, oligo dt reverse primer. dntp, 100mM DTT, Reaction buffer, MML-V reverse transcriptase (Bioneer Inc, Korea), 42 C cdna. cdna 0 min, 1 min, 5 min, 15 min, 30 min. DNA ksbv (Genbank Accession Number HQ322114) primer set DNA fragment PCR. DNA pbx pgem-3zf T-Cloning vector. DH5 F', PCR DNA (Fig. 1, Table 1). ksbv (Genbank Accession Number. HQ322114) open reading frame (ORF) capsid protein, peptidase-c3g, RNA-dependent RNA-polymerase (RdRP) primer,. URRT-PCR primer capsid protein (Fig. 1, Table 1). ksbv- primer, PCR. PCR primer plasmid 2.49 10 5 molecules, 5 pmole primer, 2x SsoFast EvaGreen Supermix (BIORAD) 20µl, real-time PCR. Realtime PCR 94 C, 10 sec, 1 823 1350 1658 2221 2549 3127 6464 7048 7685 8755 8755 Fig. 1. Locations of specific primers in Korean Sacbrood Virus genome (Genbank Accession Number HQ322114). Table 1. Oligo-nucleotide primer sets for RT-PCR from ksbv-specific target gene Primer Sequence (5' 3') Length (mer) Tm ( C) ksbv-c3f(+) GAT AGA TCT CCA GCG CGT GTT GAG TTT GTT 30 60 ksbv-c3r(+) GTA GTC GAC TAC TGG GTC CTT CCC AAT CTT 30 57 ksbv-vp1-f GCT GGA TCC TGG ATG CCT ATA AAT TCA ATT 30 49 ksbv-vp1-r GCT GTC GAC AAC TTT GTA CGA CAT TCC CGC 30 57 ksbv-urrt-f CTT ACG CTA AGT GCG CGC 18 59 ksbv-urrt-r AGC ACG ATG ATA TCT AAA GGG 21 54 ksbv-rdrp-ef CAC GGA TTC AAG CGA AAA TCC GAG TTG 27 51 ksbv-rdrp-er CCG GTC GAC TAT CCA TTG AGT GGT ATC AT 29 49 ksbv-vp2-f GAT GGA TCC ACG TTT AAT TGG GGA TCA GGC 30 57 ksbv-vp2-r GAT GTC GAC AGT TGA CTG TTT TAA ACC ATG 30 50 Product size (bp) 823 528 257 894 582

124 94 C, 5 sec, / 60 C, 5 sec 50 cycle, PCR product melting 70 C 90 C. primer set gradient RT- PCR. RT-PCR 2x SsoFast EvaGreen Supermix (BIORAD), forward, reverse primer 5 pmole, target DNA 2.49 10 5 molecules 20µl volume. Gradient real-time PCR Exicycler Quantitative Thermal Block (Bioneer), PCR 94 C 10 sec, 94 C 5 sec, / 42~70.67 C 5 sec 40 cycle, PCR Melting 70 C 90 C. ksbv-vp2 primer set, pgem-ksbv-vp2 plasmid serial dilution 2.49 10 7-1. Real- Time PCR 5 pmole ksbv-vp2 primer, 2x SsoFast EvaGreen Supermix (BIORAD). Realtime PCR 94, 10 sec, 94 C, 5 sec, / 60 C, 5 sec 50 cycle, PCR melting 70 C 90 C. Contamination test ksbv-vp2 primer test genomic DNA 1µg. RT-PCR 94 C 5 sec, 3 sec, 2 sec, / 60 C 5 sec, 4 sec, 3 sec RT-PCR. PCR melting 76 C 86 C. URRT-PCR DNApolymerase 2 PCR premix. premix 2X SsoFast EvaGreen Supermix (BIORAD) 2X fast RT-PCR premix mix, PCR. 2X SsoFast EvaGreen Supermix (BIORAD) 95 C, 5 sec, / 60 C 5 sec 2-step URRT-PCR 40 cycle, 2X fast RT-PCR premix mix 95 C 5 sec, 60 C 5 sec, 72 C, 5 sec 3-step URRT-PCR 40 cycle. ksbv-vp2 primer set URRT-PCR 95 C, 10 sec, 93 C, 94 C, 95 C, 96 C, 97 C 2 sec 60 C 40 cycle. 58 C, 59 C, 60 C, 61 C, 62 C 5 sec,, 95 40 cycle. Melting 70 C 90 C (0.5 C/sec). URRT-PCR target DNA 2.49 10 7, 10 pmole ksbv-vp2 primer 2x fast RT- PCR premix mix 6µl. ksbv-vp2 primer set pgem-ksbv-vp2 plasmid serial dilution 2.49 10 7-1. ksbv-vp2 primer set URRT-PCR 95 C, 10 sec,, 95 C, 5 sec, 60 C, 5 sec 72 C, 5 sec 40 cycle. Melting 70 C 90 C(0.5 C/sec).

125 URRT-PCR 95 C 10 sec,, 95 C 2 sec, 1 sec, 60 C 5 sec, 4 sec, 3 sec 72 C PCR. URRT-PCR target DNA 2.49 10 7, 10 pmole ksbvvp2 primer 2x fast RT-PCR premix mix 6µl. ksbv-vp2 plasmid DNA target DNA 10 5 molecules 2-step RT-PCR / 30 sec 2 sec, 3 sec PCR (Fig. 4). ksbv RNA 1 ng cdna. MML-V ksbv genome primer sets(table 1) target DNA 10 5, 10 4 RT-PCR. primer sets target DNA 10 5, primer set ksbv-urrt, ksbv-vp1, ksbv-c3-823, ksbv-vp2, ksbv-rdrp 12.96, 24.54, 22.80, 20.19, 29.37 threshold cycles (C T value) (Fig. 2 and Table 2)., ksbv-vp2 primer set RT-PCR. ksbv-vp2 plasmid serial dilution ksbv-vp2 primer set 10 1 target DNA. (Regression coefficient), R 2 0.9983 (Fig. 3). Fig. 2. Primer selection using Real-Time PCR and melting analysis of RT-PCR product. Each target gene was amplified by each primer set using Real-time PCR. ksbv-vp2 amplicon was detected as highest fluorescence more than others. Table 2. Resuts of Real-Time PCR using various primer sets and target DNA Primer Name ksbv-urrt ksbv-vp1 ksbv-c3-823 ksbv-vp2 ksbv-rdrp Molecules of target DNA 10 5 10 4 10 5 10 4 10 5 10 4 10 5 10 4 10 5 10 4 Ct value 12.96 14.39 24.54 27.23 22.80 26.27 20.19 22.77 29.37 29.88 Tm ( C) 79 79 81 80.5 82.5 82.5 80.5 81 75 75 Final fluorescence value 9630 8420 21375 16942 20117 17746 21996 21427 7277 5798

126 Fig. 3. Sensitivity test of ksbv-vp2 primer set using RT-PCR and standard curve. ksbv-vp2 primer set could detect until 10 1 molecules of target gene. It was confirmed by fluorescence graph of real-time PCR. ksbv-specific real-time PCR was defined to high quantity about quantitatively intial template. 2sec - 3sec 4sec - 5sec 5sec - 5sec 5sec - 4sec Fig. 4. Minimized time of denaturation and annealing/extension conditions were affect to real-time PCR amplicon. In realtime PCR used ksbv-vp2 primer set, even though, denaturation and annealing/extension time were decreased, Ct values were rarely changed. Final fluorescence graph were unrelated with PCR condition times. reverse transcriptase (Bioneer) cdna 42 C 1, 2, 3, 4, 5, 15, 30, 60, Fig. 5. Real-Time PCRs with ksbv-vp2 primer using various cdnas generated on various time of incubation. cdna products were synthesized by MMLV reverse transcriptase under the same conditions with different reaction times. They were performed by real-time PCR. In fluorescence graph, Ct values of 30 min reaction time and 1 min reaction time were rarely changed. It was shown that reaction time were not important factor at cdna synthesis. Actually, created cdna from 1 min reverse transcription reactions could amplify target gene by real-time PCR. cdna ksbv-vp2 primer real-time PCR (target DNA ). Real-time PCR 29.56, 29.55, 29.43, 28.75, 28.54, 28.38, 27.57, 26.14 threshold cycles (C T value) (Fig. 5, Table 3). URRT-PCR 2x SsoFast EvaGreen Supermix 2X fast RT-PCR premix mix, ksbv-vp2 primer set, target DNA

127 Table 3. Ct value of RT-PCR with ksbv-vp2 primer using various cdnas generated on various time of incubation cdna synthesis time (minute) Negative RNA 0 1 5 15 30 Ct value 35.76-29.43 27.79 26.84 27.65 28.50. URRT-PCR master mix 2x fast RT-PCR premix Ct 21.55 cycles, 2x SsoFast EvaGreen Supermix Ct 33.99 cycles, 12.44 cycles. 40 2 A 1.39 (Fig. 6). ksbv-specific URRT-PCR, ksbv-vp2 primer set, 94 C, 10 sec, 94 C, 60 C A 1.39 1.11 0.83 0.55 0.27 0 Fast taq Fast taq without DNA Ssofast Ssofast without DNA 5 10 15 20 25 30 35 40 1.11 0.83 0.55 0.27 0 94 C 95 C 93 C 96 C 97 C 5 10 15 20 25 30 35 40 B 1.04 B 1.39 0.83 1.11 0.62 0.41 0.20 Ssofast without DNA Fast taq without DNA Fast taq Ssofast 60 65 70 75 80 85 90 Dissociation (Temp) Fig. 6. Comparison between DNA-polymerase (master mix) using Ultra-rapid real-time PCR. (A) URRT-PCR fluorescence graph using ksbv-vp2 primer set, (B) Melting analysis. 2x fast RT-PCR premix was performed under the 95 C pre denaturation 1min, 94 C denaturation 5 sec, 60 C, annealing 5 sec, 72 C Extension 5 sec conditions, using ksbv-vp2 primer set. 2x SsoFast EvaGreen Supermix was performed under the 95 C pre denaturation 1 min, 94 C denaturation 5 sec, 60 C annealing/extension 5 sec conditions, using ksbvvp2 primer set. In URRT-PCR, 2x fast RT-PCR premix had 21.55 ct value, 2x SsoFast TM EvaGreen Supermix had 33.99 Ct value. 2x fast RT-PCR premix had confirmed more fast amount of 12.44 cycles than 2x SsoFast TM EvaGreen Supermix. 0.83 0.55 0.27 60 C 62 C 61 C 59 C 58 C 0 5 10 15 20 25 30 35 40 Fig. 7. Optimization of denaturation and annealing conditions using ksbv-vp2 primer set in URRT-PCR. 2.49 10 7 molecules of target DNA, 10 pmole ksbv-vp2 primer sets and 2x fast RT-PCR premix were used for URRT-PCR in total volume 6 ul. (A) Denaturation temperature was performed to 93 C, 94 C, 95 C, 96 C, 97 C under the fixed 60 C, annealing temperature. (B) Annealing temperature was performed to 58 C, 59 C, 60 C, 61 C, 62 C under the fixed 94 C, denaturation temperature. URRT-PCR denaturation and annealing conditions were defined to 94 C, 60 C, because they had shown the highest fluorescence value.

128 (Fig. 7). 0.49 ksbv-vp2 plasmid DNA 10 7-1 serial dilution. ksbv-vp2 primer ksbv-specific URRT-PCR 2.49 10 2 molecules (target DNA), A 0.79 0.63 0.47 0.31 0.15 B Ct value 30 20 10 7 10 6 10 5 10 4 10 3 10 2 0 5 10 15 20 25 30 35 40 Standard curve R 2 = 0.9928 0.39 0.29 0.19 0.09 0 5 10 15 20 25 30 35 40 Fig. 9. URRT PCR for minimizing ksbv detection time : minimized time of each cycle step. URRT-PCR was amplified under the optimum conditions, but time of each cycles step were performed differently as denaturation 95 C 3 sec, 2 sec, 1 sec, annealing 60 C 3 sec, 2 sec, 1 sec, extension 72 C 5 sec, 4 sec, 3 sec. In URRT-PCR, minimized time of each step shown that denaturation 1 sec, annealing 1 sec and extension 3 sec conditions could amplify target gene. Process of amplification has same appearance of Ct value and amplicon. Minimized times of each steps were rarely affect to total PCR reaction. In graph was indicated sec (second). URRT-PCR,,, 1 sec, 1 sec, 3 sec ksbv-vp2 Primer ksbv-specific PCR target DNA (Fig. 9). 10 0 10 7 10 6 10 5 DNA molecules number Fig. 8. Detection limit using URRT PCR with ksbv-vp2 primers. Using serial dilution of pgem-ksbv-vp2 plasmid DNA, each template of 2.49 10 7,6,5,4,3,2,1 molecules was amplified by URRT-PCR. ksbv-specific URRT-PCR was performed under optimum conditions. Detection limit of ksbv-specific URRT-PCR was 2.49 10 2 molecules, and a permitted limit of quantity was 2.49 10 7 ~2.49 10 5. ksbv-vp2 primer, 2.49 10 5 target DNA 40 URRT-PCR. 95 C 1 min, 95 C 1 sec, 60 C 1 sec, 72 C 3 sec,, 40 PCR 14 min 28 sec, 6 min 12 sec (24.86 C T ) ksbv (Fig. 10). 2.49 10 7 ~2.49 10 5 (Fig. 8). ksbv-vp2 plasmid DNA 10 7 target DNA (real-time) PCR

129 A 0.49 0.39 0.29 0.19 0.09 B 0.64 0.51 0.38 0.25 0.12 0 5 10 15 20 25 30 35 40 0 10 20 30 40 Dissociation (Temp) Fig. 10. Ultra-rapid ksbv detection using ultra-rapid real-time PCR. (A) URRT-PCR Ct graph using ksbv-vp2 primer set, (B) Melting temperature analysis, ksbv-specific URRT-PCR was performed under the optimum conditions. URRT-PCR 40 cycle was taken for 14 min 28 sec, detection of ksbv was confirmed at 6 min 12 sec in 24.86 Ct value.. PCR (Immunochromatography) (easy) (rapidity). PCR PCR, RNA-genome - PCR., RNA genome DNA cdna, ( ; MMLV-reverse transcriptase), 30 1., DNA, PCR, PCR(URRTRT-PCR) cdnageneration., Giang et al. (2015) BQCV (Black queen cell virus), 1, 5 PCR PCR., cdna 1 target DNA PCR, 30 cdna C T 0.71.,, PCR target DNA, (Fig. 5; Table 3). PCR 1, 1, 3 PCR. primer, primer (annealing ability) primer. PCR primer., PCR, PCR,, ksbv-vp2 primer PCR (cycle), (step) 3 ~5, target DNA 10 1.,

130 1 ~3 target DNA 10 5, PCR PCR (quantitative PCR)., PCR DNA-polymerase. 2X fast RT-PCR premix 2X SsoFast EvaGreen Supermix DNA (extreme thermophile DNA-dependent DNA polymerase), premix URRT-PCR. 21.55 Ct, 33.99 Ct,, URRT-PCR DNA DNA. - PCR (ksbv-specific Ultra-Rapid Fig. 11. URRT-PCR model for the most fastest detection of ksbv. Using minimized times of cdna synsthesis method and ultra-rapid real-time PCR method, UR-RT-RT PCR model for detection of Korean sacbrood virus rapidly. Using ksbv minimized detection model, detection time was totally 14 min 28 sec / 40 cycles containing cdna synthesis, melting analysis. Minimum detection time of ksbv was 7 min 12 sec (in 24.86 Ct value) containing cdna synthesis.,., (Korean Sacbrood Virus; ksbv) 2010. 2010~2015 90%,,.., PCR(URRT-PCR) cdna,. - PCR, 7 12, 2.5 10 1 ksbv DNA. ksbv-urrtrt PCR. :, PCR,,, Reverse Transcription Real-Tme PCR), cdna 1, PCR 6 12 (C T 24.86),., (

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