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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)

1 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 During the time 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 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. bsyoon@kyonggi.ac.kr; chilwoo.lee@gmail.com 121

2 122 (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 HQ ml 2 homogenizer. intron R&A-Blue

3 123, 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 molecules, 5 pmole primer, 2x SsoFast EvaGreen Supermix (BIORAD) 20µl, real-time PCR. Realtime PCR 94 C, 10 sec, 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 ksbv-c3r(+) GTA GTC GAC TAC TGG GTC CTT CCC AAT CTT ksbv-vp1-f GCT GGA TCC TGG ATG CCT ATA AAT TCA ATT ksbv-vp1-r GCT GTC GAC AAC TTT GTA CGA CAT TCC CGC ksbv-urrt-f CTT ACG CTA AGT GCG CGC ksbv-urrt-r AGC ACG ATG ATA TCT AAA GGG ksbv-rdrp-ef CAC GGA TTC AAG CGA AAA TCC GAG TTG ksbv-rdrp-er CCG GTC GAC TAT CCA TTG AGT GGT ATC AT ksbv-vp2-f GAT GGA TCC ACG TTT AAT TGG GGA TCA GGC ksbv-vp2-r GAT GTC GAC AGT TGA CTG TTT TAA ACC ATG Product size (bp)

4 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 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 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,, cycle. Melting 70 C 90 C (0.5 C/sec). URRT-PCR target DNA , 10 pmole ksbv-vp2 primer 2x fast RT- PCR premix mix 6µl. ksbv-vp2 primer set pgem-ksbv-vp2 plasmid serial dilution 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.

5 125 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 , 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, 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 (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 Ct value Tm ( C) Final fluorescence value

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.

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.

Final fluorescence graph were unrelated with PCR condition times. reverse transcriptase (Bioneer) cdna 42 C 1, 2, 3, 4, 5,

cdna products were synthesized by MMLV reverse transcriptase under the same conditions with different reaction times. They were performed by real-time PCR.

It was shown that reaction time were not important factor at cdna synthesis.

6 126 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, 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

7 127 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 Ct value URRT-PCR master mix 2x fast RT-PCR premix Ct cycles, 2x SsoFast EvaGreen Supermix Ct cycles, cycles A 1.39 (Fig. 6). ksbv-specific URRT-PCR, ksbv-vp2 primer set, 94 C, 10 sec, 94 C, 60 C A Fast taq Fast taq without DNA Ssofast Ssofast without DNA C 95 C 93 C 96 C 97 C B 1.04 B Ssofast without DNA Fast taq without DNA Fast taq Ssofast 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 ct value, 2x SsoFast TM EvaGreen Supermix had Ct value. 2x fast RT-PCR premix had confirmed more fast amount of cycles than 2x SsoFast TM EvaGreen Supermix C 62 C 61 C 59 C 58 C Fig. 7. Optimization of denaturation and annealing conditions using ksbv-vp2 primer set in URRT-PCR 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.

8 128 (Fig. 7) ksbv-vp2 plasmid DNA serial dilution. ksbv-vp2 primer ksbv-specific URRT-PCR molecules (target DNA), A B Ct value Standard curve R 2 = 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) 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 ,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 molecules, and a permitted limit of quantity was ~ ksbv-vp2 primer, 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) ~ (Fig. 8). ksbv-vp2 plasmid DNA 10 7 target DNA (real-time) PCR

9 129 A B 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 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.,

10 130 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 Ct, 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 Ct value) containing cdna synthesis.,., (Korean Sacbrood Virus; ksbv) ~ %,,.., PCR(URRT-PCR) cdna,. - PCR, 7 12, ksbv DNA. ksbv-urrtrt PCR. :, PCR,,, Reverse Transcription Real-Tme PCR), cdna 1, PCR 6 12 (C T 24.86),., (

11 , ), ( ) ( ), Bailey, L Recent research on honey bee viruses. Bee World. 56: Cho, Y.K., J.T. Kim, Y.S. Lee, Y.A. Kim, K. Namkoong, H.K. Lim, K.W. Oh, Sh.H. Kim, J.I. Han, C.S. Pak, C.S. Ki, J.R. Choi, H.K. Myeong, C. Ko Clinical evaluation of micro-scale chip-based PCR system for rapid detection of hepatitis B virus. Biosens. Bioelectron. 21: Ghosh, R.C., B.V. Ball, M.M. Willcocks and M.J. Carter The nucleotide sequence of sacbrood virus of the honey bee: an insect picornavirus. J. Gen. Virol. 80: Giang, T.H.L., J.S. Lee, S.J. Yong and B.S. Yoon Development of ultra-rapid reverse transcription realtime PCR for detection against Black queen cell virus in honeybee. J. Apiculture 30(3): Han, S.H., Y.S. Choi and M.L. Lee Development of highly specific quantative real-time PCR method for the detection of Sacbrood virus in Korean honeybees, Apis cerana. J. Apiculture 26(3): Kang, M.H., I.W. Kim, D.W. Lee, M.S. Yoo, S.H. Han, B.S. Yoon Development of a rapid detection method to detect tdh gene in Vibrio parahaemolyticus using 2-step ultrarapid real-time polymerase chain reaction. Diagnostic Microbiology and Infectious Disease 69: Kim I.W., M.H. Kang, S.H. Kwon, S.H. Cho, B.S. Yoo, S.H. Han, B.S. Yoon Rapid detecton of virulence stx2 gene of Enterohemorrhagic Escherichia coli using twostep ultra-rapid real-time PCR. Biotechnology Letters. 32: Lee, D.W., E.H. Kim, I.U. Kim, Yoo M.S., Yoon BS Ultra-rapid two-step real-time PCR for the detection of human immunodeficiency virus (HIV). Kor J Microbiol. 43: Ma, M., Ma, C., Li, M., Wang, S., Yang, S, Wang, S Loop-mediated isothermal amplification for rapid detection of Chinese sacbrood virus. Journal of virological methods. 176(1):

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Æ¯ÇãÃ» '' - 1 - - 2 - - 3 - - 4 - - 5 - ' - 6 - - 7 - - 8 - - 9 - - 10 - - 11 - Super-Bio Co., Ltd. KWON, Suk-Tae Thermostable DNA Polymerase-encoding Gene from Thermus sp. X-1 an d Amino Acid Sequence