Original Article Journal of Apiculture 31() : 133~1 (1) Development of a Detection Method against 11 Major Pathogens of Honey Bee using Amplification of Multiplex PCR and Specific DNA-chip Ji-Hee Wang, Dobu Lee 1, Su-Jin Ku 1, Mun-Cheol Peak 1 *, Sang-Hyun Min, Su-jin Lim, Chil-Woo Lee and Byoung-Su Yoon* Department of Life Science, Kyonggi University, Suwon 17, Korea 1 K-MAC, 33, Techno -ro, Yuseong-gu, Daejeon, 3, Korea (Received 19 June 1; Revised 3 June 1; Accepted 3 June 1) Abstract Against 11 major honeybee pathogens, new detection-method using multiplex PCR and specific DNA-chip were developed. 11 major pathogens were species of bacteria, 3 of fungi and of viruses, including Paenibacillus larvae, Melissococcus plutonius, Aspergillus flavus, Ascosphaera apis, Nosema ceranae, Black queen cell virus, Chronic bee paralysis virus, Deformed wing virus, Israeli acute paralysis virus, Sacbrood virus, and Korean sacbrood virus. Development of multiplex PCR and DNA-chip were well optimized just using DNA and cdna isolated from a infected sample, such as larva or adult honeybee. Based on analysis of DNA chip, it could be easy recognized, which are dominant among 11 pathogens in test sample. It might be useful as confirmation-test for diagnosis of honeybee diseases. Key words: Multiplex PCR, DNA chip, Honey bee diseases, Qualitative detection, Confirmation test,, 35 (Chen and Siede, 7; Runckel et al., 11; Li et al., 1), 15. 11,. 11, (American Foulbrood *Co-corresponding author. E-mail: bsyoon@kgu.ac.kr; mcpaek@kmac.com 133
13 disease; AFB), Paenibacillus larvae, (European Foulbrood disease; EFB) Melissococcus plutonius, (Chalkbrood; CB) Ascosphaera apis, (Stonebrood; SB) Aspergillus flavus Nosema ceranae 3. BQCV(Black Queen Cell Virus), CBPV(Chronic Bee Paralysis Virus) DWV (Deformed Wing Virus; ), IAPV(Israeli Acute Paralysis Virus), SBV(Sacbrood Virus), ksbv(korean Sacbrood Virus; ). ksbv SBV 75% (Choi et al., ). Nosema ceranae Nosema apis. 11, PCR, (Carletto et al., ;, 1).,, PCR(quantitative PCR) (Han et al., 11; Bu et al., 5), PCR(Ultra-rapid PCR) (Han et al., ; Yoo et al., 11, ; Lim and Yoon, 13), Ultra-fast PCR (UF- PCR) LAMP (Loop-mediated amplification) (, 1; Lee et al., 1)., (Immunochromatography), (Unpublished communication).,,,. ( ) multiplex PCR, Sguazza (13) ABPV, BQCV, CBPV, DWV, IAPV, SBV multiplex PCR. multiplex PCR PCR,., DNA chip oligonucleotide(probe) slide glass (microarray) in situ (spotting), DNA probe DNA:DNA hybridization (Southern et al., 199)., PCR, probe DNA-chip hybridization, PCR hybridization,, probe, ( ) (Sarshar et al., 15; Li, 1).,, DNA chip.,,,, 11 multiplex PCR, PCR, probe DNA-chip. 1 5 ksbv.,
135 MagNa lyser(roche, Korea) RNA iso plus(takara, Japan) total RNA. RNA spectrophotometer, 1 µg unit M-MLV reverse transcriptase (Bioneer, Korea) cdna, cdna total RNA 7 C. PCR DNA,. DNA, (Table 1). 11 PCR primer 11, primer multiplex PCR, PCR DNA-chip probe (oligonucleotides) hybridization, Table 1. primer, multiplex PCR Real-Time PCR, primer,,, DNA-chip probe. DNA-chip probe oligonucleotide hybridization PCR primer 11 primer 5 Cy3 (Bioneer, Korea), Primer Real-Time PCR SyberGreen. Real-Time PCR Exicycler 9(Bioneer, Korea). Table 1. The recombinant DNAs of honey bee pathogens Pathogens Name of clone GenBank accession no. Reference BQCV pgem-bqcv-rdrp EF517515 Giang et al., 1 CBPV AM-CBPV EU31, DWV plug-dwv-rdrp N, 15 IAPV pdrive-iapv-rdrp NC95 Unpublished SBV pbx-sbv3 AF99 Kim et al., ksbv pgem-ksbv-vp1 HQ311 Unpublished Paenibacillus larvae pbx-p.la-1s-797 U53 Unpublished Melissococcus plutonius pdrive-efb X75751, 5 Ascosphaera apis pbx-a.apis M3, Aspergillus flavus pbx-a.flavus D39 Lee et al., 15 Nosema ceranae pcr.1-nosema DQ7 This study Table. Origins and names of primer sets for multiplex PCR Pathogens Name of clone GenBank accession no. Reference BQCV BQ-PCR-F1/R1 EF517515 This study CBPV CB-PCR-F1/R1 EU31 This study DWV DW-PCR-F1/R1 N This study IAPV KBIA-PCR-F/R NC95 This study SBV SB-PCR-F1/R1 AF99 This study ksbv ksb-pcr-f1/r1 HQ311 Unpublished Paenibacillus larvae AFB-PCR-F1/R1 U53 This study Melissococcus plutonius EFB-PCR-F1/R1 X75751 This study Ascosphaera apis AA-PCR-F1/R1 M3 This study Aspergillus flavus AF-PCR-F1/R1 D39 This study Nosema ceranae NO-PCR-F/R1 DQ7 This study
13 11 DNA, DNA copies 5., DNA PCR primer (annealing) PCR(temperature-gradient PCR),, -multiplex PCR. PCR µl, PCR premix Greenstar master mix (Bioneer, Korea). 11 DNA -multiplex PCR PCR cycle, - multiplex PCR. -multiplex PCR -Asy-primer mix, 9 C 5, 9 C 3, 5 C 3, 7 C 3 35 cycles. -Asy-primer mix, PCR DNA-chip probe oligonucleotide hybridization, (asymetric) PCR primer primer-mixture., primer 1: :1 1:(forward : reverse primer ) hybridization SBR(Spot/Background Ratio), -asy-primr mix. multiplex-pcr PCR hybridization, hybridization,. DNA-chip K-MAC(Korea), hybridization K-MAC K-SCAN- CAP scanner(k-mac, Korea) software program(k-mac, Korea). K-CAP K-Mac DNA-chip, µl. PCR, probe oligonucleotide (Fig. 1) 11 probe oligonucleotide, probe pmole/µl pmole/µl, probe sciflexarrayer S11(Scienion, Germany), aldehyde K- CAP pl (spotting). probe µm spot, (center to center; CTC) 1µm. Probe 5 C, 7% 3 K-CAP aldehyde probe amine, Prehybridization Buffer(x SSPE,.% SDS) probe (K-MAC, Korea) DNA-chip oligonucleotide probe PCR, hybridization (Bioneer, Korea). oligonucleotide probe DNA-chip, K-CAP hybridization, Fig. 1. K-CAP TM, DNA chip for detection of honey bee pathogens manufactured by K-MAC, Korea.
137 -Asy primer mix multiplex PCR, PCR PCR (hybridization). 95 C 5, 5 C, 3 rpm (Thermo-mixer comfort, Eppendorf, Germany), (washing) SSC buffer,. SSC buffer, D.W. 3 rpm C 5. 7 C, 5 C, C, (xssc, xssc). spot SBR(Spot/Background Ratio). 11 5 ( pg), primer set PCR primer - Asy-primer mix( pmole/reaction) primer P. pmole/reaction. PCR µl, Greenstar master mix(bioneer, Korea) multiplex PCR. Multiplex PCR, 9 C 5, 9 C 3, 5 C 3, 7 C 3 cycles, hybridization 5 C, 3 rpm, washing C, 3 rpm.5 ml SSC buffer, 5,.5 ml. SSC buffer, 5,.5 ml D.W. 5, LightCycler Multi-well-plate 9, white(roche, Korea) 3,5 rpm, DNA-chip. Hybridization K-SCAN-CAP, SBR (Spot/Background Ratio). ksbv total RNA, 1µg µl cdna, 1µl -Asy primer mix multiplex PCR DNA-chip hybridization..,, DNA-chip cdna-target ksbv-specific Real-Time PCR target. Real-Time PCR primer -Asy primer mix ksb-pcr-f1/r1, Real-Time PCR 5mM ksb-pcr-f1/r1 PCR premix Greenstar master mix(bioneer, Korea), multiplex PCR 1µl ksbv cdna. Real-Time PCR 9 C 5, 9 C 3, 5 C 3, 7 C 3 cycles. pgem-ksbv-vp1 DNA target, 5,, 3,. PCR, multiplex PCR annealing, DNA 5, primer 5 pmole, (temperature of mid-point; Tm) PCR (Fig. ). 11 primer PCR, (melting temperature analysis) Tm. 11 PCR
13, PCR annealing 5 C, multiplex PCR annealing ( ). A Fig. 3. Multiplex PCR using -Asy primer mix with each pathogen-specific DNA. As template, each 5 molecules of pathogen-specific DNA were added. -Asy primer mix were used to each PCR. The Ct values of BQCV, CBPV, DWV, SBV, AFB, EFB, Ascosphaera apis, Aspergillus flavus and Nosema ceranae were estimated.~3. cycles, except. cycles of IAPV and ksbv. All final fluorescence intensities were pointed on 3.3~5. K. B Fig.. The amplification of M. plutonius (EFB)-specific DNA with specific primers using annealing temperature-gradient PCRs. (A) Fluorescence curves. M. plutonius-specific PCR products were differently amplified depend on annealing temperature, C, C, 5 C and 5. (B) Melting-temperature analysis. The Tms of M. plutonius-specific PCR products were estimated in range of 7.5~9. C 11 specific multiplex-pcr, 11 ( ) primer 1 pmole pmole primer, DNA 5., primer (binding affinity), 11 PCR product, primer, primer PCR, DNA-chip probe primer-mix. DNA-chip multiplex PCR premix, -Asy primer mix( pmole/ each PCR) (Table 3). DNA 5, -Asy primer mix multiplex PCR, DNA, 9 target DNA( 5 ) Ct(Threshold cycles) -3 Table 3. Composition of -Asy primer mix for pathogen specific multiplex-pcr & DNA-chip Targets Template ( pg) Name of primer Forward primer/rxn Reverse primer/rxn BQCV.55 5 copies BQ-PCR-F1/R1 pmole 1 pmole CBPV.9 5 copies CB-PCR-F1/R1 1 pmole pmole DWV 5.1 5 copies DW-PCR-F1/R1 1 pmole pmole IAPV 3. 5 copies KBIA-PCR-F/R pmole pmole SBV.9 5 copies SB-PCR-F1/R1 1 pmole pmole ksbv.99 copies ksb-pcr-f1/r1 1 pmole pmole P. larvae.9 5 copies AFB-PCR-F1/R1 1 pmole pmole M. plutonius. 5 copies EFB-PCR-F1/R1 1 pmole pmole Ascosphaera apis. 5 copies AA-PCR-F1/R1 pmole 1 pmole Aspergillus flavus. copies AF-PCR-F1/R1 1 pmole pmole N. ceranae. 5 copies NO-PCR-F/R1 1 pmole pmole Total primer quantity/reaction: pmole/µl rxn=reaction
139 cycles (, ksbv IAPV Ct cycles)., 11 (final fluorescence intensity) 3.3~5. K, hybridization (Fig. 3) DNA chip K-CAP (K-MAC, Korea),,.ml PCR tube PCR DNA. Tube DNA.mm, 7, 7, 9 oligonucleotide probe spot, spot.mm, spot (Center to Center).1mm. probe oligonucleotede, probe spotting DNA-chip, spot Fig.. Positions of each spot containing oligonucleotide probes on DNA chip, K-CAP TM. 19 different oligonucleotide probes were mounted on spots in each set. Different quantities of oligonucleotide probes were used on spots in Set 1 ( pmole) and on spots in Set ( pmole), positions of Hybridization control (HC) were located. (HC; hybridization control). spot Set 1 19, Set 19, Table. Positions and names of oligonucleotide probes on DNA chip, K-CAP TM Spot site Probe Probe concentration / spot Set 1 Set oligonucleotides Set 1 (pmole) Set (pmole) Genbank accession no. B1 BQCV-PP-R EF517515 B E CBPV-PP1-F EU31 B3 DWV-PP-F N B E DWV-PP-F N B5 IAPV-PP5-F NC95 B E IAPV-PP-R NC95 B7 Apis-PP-R D39 F7 Apis-PP5-R D39 F1 SBV-SPP3-F AF99 C F SBV-SPP5-F AF99 F3 SBV-SPP-F AF99 C F ksbv-pp1-f HQ311 F5 ksbv-pp3-f HQ311 C F Nosema-PP1-F U53 D1 AFB-PP1-F X75751 D G AFB-PP3-F X75751 D3 EFB-PP1-F M3 D G EFB-PP3-F M3 D G flavus-pp3-r DQ7 Total probe oligonucleotides 19
1, 11 probe 19, Set 1 pmole spotting, Set pmole spotting, DNA-chip (Fig. ). 11 oligonucleotide probe 19 probe. SBV ksbv 3, probe spotting, DWV, IAPV, AFB, EFB probe, BQCV, CBPV, Nosema cerana, Ascosphaera apis, Aspergillus fluvus 1 probe (Table ). Paenibacillus larvae(afb), Melissococcus plutonius (EFB), Nosema ceranae, Ascosphaera apis, Aspergillus fluvus, SBV, ksbv, DWV, IAPV, BQCV, CBPV 11 DNA. 5 DNA PCR, -Asy primer mix PCR, DNA-chip. -Asy primer mix, multiplex PCR DNA-chip AFB target asy multi mix (hrs hybrid) B B B D D D F F F BG BG Fig. 5. The fluorescence image and signal intensity of Paenibacillus larvae detection using -Asy primer mix and target DNA. The.9 5 molecules of AFB-specific DNA were used and -Asy primer mix and HC primer were added. The AFB specific D1 (probe set 1, pmole) and (Probe set, pmole) were 9.3. D (probe set 1, pmole) and G (probe set, pmole) were 5. and 5.3. HC sites were,, A, and. EFB target asy multi mix (hrs hybrid) B B B D D D F F F BG BG Fig.. The fluorescence image and signal intensity of Melissococcus plutonius detection using -Asy primer mix and target DNA. The EFB specific D3 (probe set 1, pmole), D (probe set 1, pmole) were.95 and 9.. The SBR value of (probe set, pmole), and G (probe set, pmole) were.5, and..
11 9 7 5 3 1 A.apis target asy multi mix (hrs hybrid) B B B D D D F F F BG BG Fig. 7. The fluorescence image and signal intensity of Ascosphaera apis detection using -Asy primer mix and target DNA. All B7,, and F7 of Ascosphaera apis specific probes showed.99 on SBR value. B7 and contain probe set 1 ( pmole). and F7 contain probe set ( pmole). A.flavus target asy multi mix (hrs hybrid) B B B D D D F F F BG BG Fig.. The fluorescence image and signal intensity of Aspergillus flavus detection using -Asy primer mix and target DNA. The Aspergillus flavus specific probe D (probe set 1, pmole) and G (probe set, pmole) were.91 and 3.3. Nosema target asy multi mix (hrs hybrid) B B B D D D F F F BG BG Fig. 9. The fluorescence image and signal intensity of Nosema ceranae detection using -Asy primer mix and target DNA. Both specific probes C (probe set 1, pmole) and F (probe set, pmole) of Nosema ceranae were.9. probe hybridization, PCR (Fig. 5,, 7,, 9,, 11,, 13, 1, 15). ksbv
1 BQCV target asy multi mix (hrs hybrid) B B B D D D F F F BG BG Fig.. The fluorescence image and signal intensity of BQCV detection using -Asy primer mix and target DNA. The (probe set, pmole) of BQCV specific probe showed 7.97 on SBR value (B1 did not match up with grid in software program). CBPV target asy multi mix (hrs hybrid) B B B D D D F F F BG BG Fig. 11. The fluorescence image and signal intensity of CBPV detection using -Asy primer mix and target DNA. The B (probe set 1, pmole) and E (Set, pmole) of CBPV specific probe showed.93 and.5. DWV target asy multi mix (hrs hybrid) B B B D D D F F F BG BG Fig.. The fluorescence image and signal intensity of DWV detection using -Asy primer mix and target DNA. The and E of DWV in probe set (Set, pmole) showed.5 and.13 (B3 and B did not match up with grid).. (Apis cerana), ksbv- PCR ksbv. PCR,, 1.97, -Asy primer mix PCR ksbv
13 IAPV targer asy multi mix (hrs hybrid) B B B D D D F F F BG BG Fig. 13. The fluorescence image and signal intensity of IAPV detection using -Asy primer mix and target DNA. Both B5 (probe set 1, pmole) and (probe set, pmole) value of IAPV specific probes were 11.. SBV target asy multi mix (hrs hybrid) B B B D D D F F F BG BG Fig. 1. The fluorescence image and signal intensity of SBV detection using -Asy primer mix and target DNA. The SBV specific C and in probe set 1 ( pmole) were 3. and 11.1. F1, F, and F3 in porbe set were 9.79,.13, and.7. ksbv target asy multi mix (hrs hybrid) B B B D D D F F F BG BG Fig. 15. The fluorescence image and signal intensity of ksbv detection using -Asy primer mix and target DNA. The ksbv specific (probe set 1, pmole) and F5 (probe set, pmole) were.1 and 5.3.. DNA-chip 1 SSC buffer DNA-chip. DNA-chip ksbv probe (C,, F, F5) PCR, SBV probe (, C,, F1, F, F3)
1 ksbv detection in honey bee sample B B B D D D F F F BG BG Fig. 1. The application of DNA chip detection using ksbv-infected honey bee sample. cdna was used 1µl (total µl cdna generation). The C, F, and F5 of ksbv specific probes showed.5,.,.5, and 5.9 on SBR value.., SBV probe (, F3) SBV probe (, C, F1, F), SBV DNA-chip., DNAchip, ksbv- PCR, ksbv (Fig. 1). DNA-chip multiplex PCR DNA chip oligonucleotide. hybridization hybridization,,. hybridization 5 C, x SSC buffer,., asymmetric primer hybridization hybridization. asymmetric primer, 3. SBR 11 -Asy primer mix. hybridization 1 hybridization 117.51%, 391.5%, hybridization, hybridization 1 hybridization. DNA-chip oligonucleotide probe,,, oligonucleotide multiplex PCR.,, multiplex PCR. multiplex PCR DNA-chip,., CBPV SBV probe (Fig. 11) Aspergillus flavus Asocsphaera apis probe (Fig. )., CBPV probe (B, E) SBV probe (, C,, F1, F, F3) CBPV SBV, Aspergillus flavus, Ascosphaera apis probe B7,, F7 Aspergillus flavus Ascosphaera apis
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