Development of methods for accurate detection of honeybee pathogens and molecular determination of adulterated honey

List of Tables Composition of Luria-Bertani liquid medium ···6 Composition of Luria-Bertani 1% agar media ···6 Primers used for SBV detection from honeybee samples and genotyping PCR ·

Thesis for Degree of Doctor

Development of methods for accurate detection of honeybee pathogens and

molecular determination of

adulterated honey

by Truong A Tai

Department of Life Science

Graduate School Kyonggi University

Development of methods for accurate detection of honeybee pathogens and

molecular determination of

adulterated honey

A dissertation submitted to the faculty of Graduate School of Kyonggi University in the fulfillment of the requirement for the Degree of Doctor of Philosophy

December 2019

Graduate School of Kyonggi University

Department of Life Science

Truong A Tai

List of Tables ···x

List of Figures ···xii

Abstract ···xvi

Part 1 Molecular characterization of honeybee pathogens ··· 1

Chapter 1 Genotyping quantification of Sacbrood virus ···1

I Introduction ···1

1 Sacbrood virus (SBV) ···1

2 Genotypes of Sacbrood virus ···2

3 Detection of SBV ···3

4 Purpose of this study ···3

II Materials and methods ···4

1 Bacterial strain and plasmid vector for molecular cloning 4 2 Bacterial culture medium ···5

3 SBV-infected honeybee samples ···6

4 Plasmid DNA isolation ···7

5 Total RNA isolation and standard DNA construction ···7

6 Primer design ···8

7 Reverse transcription ···11

8 Standard DNA construction ···12

9 Specific identification of genotyping DNA ···13

10 Sensitivity of SBV detection ···13

11 Quantitative detection of SBV genotypes ···14

12 Agarose gel electrophoresis ···15

III Results and discussion ···15

1 Standard DNAs for SBV genotyping ···15

2 Sensitivity of genotyping in single PCR and nested PCR 17 3 Accuracy of SBV genotyping on standard DNAs ···19

4 Detection of SBV genotypes from honeybee samples ···21

5 Quantification of SBV genotypes ···23

IV Conclusion ···24

Chapter 2 Evaluation of point mutation on the minor capsid protein (MiCP) gene of Sacbrood virus ···26

I Introduction ···26

II Materials and methods ···27

1 Source of SBV nucleic acids ···27

2 Detection of SBV ···27

3 Molecular cloning ···27

4 Determination of DNA sequences from each sample ··· 28

5 Analysis of SBV-specific DNA sequences ···33

III Results and discussion ···35

1 Genome of SBVs belong to two different genotypes ···35

2 Single nucleotide polymorphisms identification in analyzed sequences ···35

3 Phylogeny on SNP patterns in genotype 2100D0 ···41

IV Conclusion ···43

Chapter 3 Rapid detection of Israeli acute paralysis virus using multi-point ultra-rapid real-time PCR ···45

I Introduction ···45

II Materials and methods ···47

1 Honeybee samples ···47

2 Primer design ···47

3 Construction of recombinant DNAs ···49

4 RNA extraction ···51

5 Multi-point PCR ···51

6 Limit of detection ···52

7 Assessment of multi-point UR-qPCR efficiency ···53

8 Sequence analysis ···53

III Results and discussion ···54

1 Comparison of single- and multi-point PCR ···54

2 Sequence analysis ···59

3 Optimization of UR-qPCR ···61

IV Conclusion ···66

Chapter 4 Quantitative detection and evaluation of Melissococcus plutonius infection in honey bee ···67

I Introduction ···67

II Materials and methods ···68

1 Bacterial strain ···68

2 Cultivation of M plutonius···68

3 Microscopic enumeration ···70

4 Plate count ···71

5 DNA extraction ···72

6 PCR performance ···72

7 Relationship between microscopic and PCR-based count ··73

8 Artificial infection of M plutonius to honeybee larvae ··· 74

III Results and discussion ···75

1 Quantification of M plutonius by microscopy and plate

count ···75

2 Molecular quantification of M plutonius using qPCR ··· 76

3 Relationship between microscopic count and molecular count ···77

4 Artificial infection of M plutonius on honeybee larvae ···· 79

IV Conclusion ···84

Chapter 5 Evaluation of microscopic and molecular quantitative detection of Nosema ceranae in honeybees ···86

I Introduction ···86

II Materials and methods ···87

1 Sample preparation ···87

2 DNA extraction ···89

3 Standard DNA and N ceranae-specific primers ···89

4 Microscopic enumeration of Nosema spore ···90

5 Molecular enumeration by quantitative PCR ···90

6 Limit of Nosema detection by microscopic and molecular method ···91

7 Evaluation of N ceranae development in caged honeybees ···93

III Results and discussion ···94

1 Standard linear regression of UR-qPCR for quantification

of N ceranae···94

2 Quantification of N ceranae in caged bees fed by Bee happy solution ···96

3 Impact of Bee happy solution on honeybee ···98

4 Relationship between microscopic count and molecular count ···100

5 Limit detection of N ceranae spores by microscopy ···103

6 Limit detection of N ceranae using UR-qPCR ···105

IV Conclusion ···106

Chapter 6 Generation of monoclonal antibody for detection of N ceranae ···107

I Introduction ···107

II Materials and methods ···108

1 Preparation of pure Nosema ceranae spore ···108

2 N ceranae confirmation by specific PCR ···109

3 Antigen preparation ···110

4 Spore lysates ···110

5 Bradford assay ···111

6 Mouse immunization ···111

7 Enzyme-linked Immunosorbent Assay (ELISA) ···112

8 Myeloma cell culture ···114

9 Hybridoma generation ···114

10 Selection of successful hybridoma cell line ···117

11 Production of monoclonal antibody ascites fluid ···118

12 Western blotting ···119

13 Dot blotting ···122

III Results and discussion ···123

1 Nesema ceranae confirmation from purified spore ··· 123

2 Immunization of mice using N ceranae antigen ···124

3 Selection of hybridoma for anti-N ceranae monoclonal antibody generation ···125

4 Confirmation of anti-N ceranae monoclonal antibody ···127

5 Production of monoclonal antibody in ascitic fluid ···128

IV Conclusion ···130

Part 2 Molecular determination of adulterated honey ···131

Chapter 1 DNA identification of corn syrup adulterated honey ···131

I Introduction ···131

II Materials and method ···133

1 Maize samples ···133

2 Corn syrup sample ···133

3 Honey samples ···133

4 Production of adulterated honey ···134

5 Specific primers for detection of Maize DNA ···134

6 DNA isolation from leaf and seed of Maize ···135

7 Isolation of pollen DNA ···135

8 Purification of residual DNA ···136

9 PCR performance ···137

III Results and discussion ···138

1 Specific amplification of Maize DNA ···138

2 Maize DNA detection in natural and adulterated honeys 140 3 Comparison of residual DNA and pollen DNA in natural honeys ···143

4 Determination of corn syrup adulteration in commercial honeys ···144

5 Quantity of DNA compositions in different parts of Maize plant and in corn syrup ···147

IV Conclusion ···149

Chapter 2 Molecular identification of monofloral honey by specific quantification of typical plant compositions ···150

I Introduction ···150

II Materials and methods ···151

1 Selection of major nectar plants ···151

2 Honey samples ···152

3 Primer design and standard DNA construction ···152

4 DNA extraction ···153

5 PCR performance ···154

6 Standard curves for calculation of DNA copy ···155

III Results and discussion ···155

1 Standard linear regression for DNA quantification ···551

2 Determination of plant compositions in natural honeys ··· 156

3 Confirmation of commercial monofloral honeys ···158

IV Conclusion ···162

References ···163

Appendix ···184

Abstract in Korean (국문요지) ···190

List of Tables

<Table 1> Composition of Luria-Bertani liquid medium ···6

<Table 2> Composition of Luria-Bertani 1% agar media ···6

<Table 3> Primers used for SBV detection from honeybee samples

and genotyping PCR ···9

<Table 4> Chemical synthesized oligo nucleotides used for standard

DNA construction ···13

<Table 5> Composition of TAE buffer ···15

<Table 6> Standard sequences of 5 SBV genotypes ···17

<Table 7> Comparison of sensitivity of SBV genotyping in nested

PCR and single PCR ···18

<Table 8> T7 and M13-20R direction sequencing data of sample 1 29

<Table 9> T7 and M13-20R direction sequencing data of sample 2 30

<Table 10> T7 and M13-20R direction sequencing data of sample 3

···31

<Table 11> T7 and M13-20R direction sequencing data of sample 4

···32

<Table 12> Primers designed for Israeli acute paralysis virus (IAPV)

and Kashmir bee virus (KBV) ···49

<Table 13> Primer sequences for construction of IAPV recombinant

detection of Israeli acute paralysis virus in honeybees 55

<Table 16> KHBHI liquid medium ···70

<Table 17> KHBHI agar media ···70

<Table 18> Primer for M plutonius detection ···73

<Table 19> Quantitative result of M plutonius by microscopy and qPCR ···79

<Table 20> Measurement of N ceranae in caged bees that were fed by Bee happy solutions ···102

<Table 21> Specific primers for Nosema ceranae and Nosema apis detection ···110

<Table 22> Reagents for ELISA ···113

<Table 23> Mediums for Myeloma cell culture and selection of hybridoma ···117

<Table 24> SDS-PAGE gel preparation ···121

<Table 25> Buffer for SDS-PAGE ···121

<Table 26> Buffer for Western blot ···122

<Table 27> Primers for Maize DNAs detection ···135

<Table 28> Sequencing result of seven commercial honeys ···145

<Table 29> Quantity of Maize gDNA in honey samples ···147

<Table 30> Collection of natural honeys for nectar sources identification ···152

<Table 31> Primers for detection of seasonal nectar plants ···153

<Table 32> Detection of seasonal nectar plants from honey samples ···157

<Table 33> Plant composition detected in the commercial monofloral honeys ···160

List of Figures

<Figure 1> Genetic map of pBlueXcm vector ···5

<Figure 2> The alignment of SBV genomes shows various numbers

of missing nucleotide among the 5 genotypes from whichthe specific genotyping primers were designed ···10

<Figure 3> Schematic diagram shows primer pairs and amplicon size

of genotyping detection ···11

<Figure 4> Schematic diagram shows the standard DNAs of 5 SBV

genotypes ···16

<Figure 5> Fluorescent curves show the specific detection of

genotyping primers on standard DNAs ···20

<Figure 6> Fluorescent curves show the positive detection and

genotype identification of SBV in infected honeybeesamples ···22

<Figure 7> Compositions of SBV genotypes in honeybee samples ·· 24

<Figure 8> SBV genotyping group of 16 sequences from 4

genotype 2134D51 ···40

<Figure 13> Phylogenetic tree of 6 SNP patterns in genotype 2100D0

···43

<Figure 14> Alignment of IAPV, KBV, and ABPV DNA for design

of IAPV specific primers ···48

<Figure 15> Locations of the primers and standard DNAs used in

the present study ···48

<Figure 16> Detection of Israeli acute paralysis virus using

multi-point PCR ···56

<Figure 17> Calculation of Israeli acute paralysis virus (IAPV) cDNA

copy number in honeybee samples ···58

<Figure 18> Detection of Israeli acute paralysis virus (IAPV) and

Kashmir bee virus (KBV) in co-infected honeybees ···· 60

<Figure 19> Sequence alignment of target Israeli acute paralysis

virus (IAPV) genes ···60

<Figure 20> Process of time optimization for IAPV detection using 3

primer pairs ···62

<Figure 21> Evaluation of the sensitivity of IAPV specific primers

used in multi-point PCR at detection conditions ···63

<Figure 22> Evaluation of the sensitivity of IAPV specific primers

used in multi-point PCR at time-saving conditions ···64

<Figure 23> Correlation between reverse-transcription (RT) time and

detection time in RT-UR-qPCR ···65

<Figure 24> Observation of M plutonius under 1000 × magnification

of a light microscope ···71

<Figure 25> Observation of M plutonius under light microscope and

bacterial colony on agar plate ···76

<Figure 26> Standard curves for M plutonius calculation ···77

<Figure 27> Relationship between molecular count and microscopic

count for the quantification of M plutonius···78

<Figure 28> Detection of M plutonius from infected larvae and

pupae ···81

<Figure 29> Artificial infection of M plutonius to honeybee larvae 83

<Figure 30> Percentage of surviving larvae that were exposed to M

plutonius ···84

<Figure 31> Serial dilution of DNA and spore solutions ···92

<Figure 32> Standard curves for Nosema ceranae calculation were

created from amplification using standard recombinantDNA ···95

<Figure 33> Quantification of N ceranae in honeybees that were fed

by various concentration of Bee happy solution ···97

<Figure 34> Mortality rate of honeybees that were fed by different

concentrations of feeding solution ···99

<Figure 35> Amount of solution consumed by caged bees during the

feeding period ···100

<Figure 36> Microscopic count of purified Nosema ceranae spores104

<Figure 37> Nosema ceranae detection in serially diluted DNA

solutions ···105

<Figure 38> Immunization period and schedule of ELISA test for

anti-N ceranae antibody generation ···112

<Figure 39> Amplification curves shows the presence of only N

ceranae in the purified spores ···124

<Figure 40> Result of ELISA showed the increase of anti-N

ceranae antibody in the immunized mouse ···125

<Figure 41> Screening for hybridoma cell line that produces specific

anti-N ceranae monoclonal antibody ···126

<Figure 42> Dot-blot and western-blot show the specific detection of

N ceranae using monoclonal antibody ···128

<Figure 43> Result of ELISA showed the detection of N ceranae

using monoclonal antibody from ascites and cell culturalmedium ···130

<Figure 44> Amplification of genomic, chloroplast, and mitochondria

DNA from Maize samples ···139

<Figure 45> Quantification of Maize gDNA in natural and adulterated

honeys ···141

<Figure 46> Amplification of Maize DNA from natural honey and

adulterated honeys ···142

<Figure 47> Quantification of Maize mtDNA and cpDNA in natural

and adulterated honeys ···143

<Figure 48> Comparison of residual DNA to pollen DNA from

natural honeys ···144

<Figure 49> Detection of Maize gDNA in commercial honeys ···146

<Figure 50> Comparison of genomic, mitochondria, and chloroplast

DNA among different parts of Maize plant and cornsyrup ···148

<Figure 51> DNA quantity of targeted plants in honey samples ··· 158

<Figure 52> Confirmative detection of nectar plants in commercial

honeys ···161

<Figure 53> DNA quantity of plant compositions in commercial

honeys ···162

The pathogens are principal factors on the collapse of honeybeecolony worldwide, and cause of decrease of honey productivity.Therefore, the efficient technologies for accurate and rapid detection

of the pathogens are the major concern of researchers andbeekeepers The aim of this study is to develop and evaluate themolecular measures for diagnosis of viral pathogens (Sacbrood virusand Israeli acute paralysis virus), bacterial pathogen (Melissococcusplutonius), and fungal pathogen (Nosema ceranae) Furthermore,molecular differentiation was also applied to detect adulterated honeymixed with sugar syrup or unidentified its origin

Sacbrood virus (SBV) is one of the prevalent pathogen that causeshigh mortality of honeybee worker larvae The worldwide distributionand geographical genotypes of SBV have been proposed by severalscientists An Ultra-rapid real time PCR (UR-qPCR) system wasdeveloped for the fast detection and quantitative determination of eachSBV genotype in infected honeybee from this study Designation ofspecific primer-pairs and construction of standard DNAs for 5genotypes were carried out The detection for SBV genotypes wasoptimized using each standard DNA and specific primer-pairs ThenPCR system for SBV-genotyping was applied with SBV-infectedhoneybee samples SBV genotyping in 10 honeybee samples, A.mellifera, showed that 4 samples were infected by both genotypes,SBVD0 and SBVD51 Meanwhile, two samples had only genotypeSBVD0, and the other 4 samples were positive with only SBVD51

The quantitative result showed that in samples infected by singlegenotype, DNA copy of SBVD0 varied from 4.33×103 - 1.39×105

copies, and of SBVD51 were from 9.30×103 - 8.04×104 copies per 100

ng of total RNA In the samples infected by both genotypes,molecular number of SBVD0 showed a dominance over SBVD51, itvaried from 4.96×101 -2.51×105 copies and 1.67×100 - 3.74×104 copiesper 100 ng total RNA, respectively Furthermore, this quantitativenested UR PCR showed 10-fold more sensitive than single PCR fordetection of SBV genotypes

The minor capsid protein (MiCP) gene of SBV was demonstrated

to facilitate the infection of SBV to its host To understand thefrequency of mutation occur in this gene, the sequence analysis fromnumbers of SBV strains was conducted SBV-specific DNAfragments were cloned and sequenced by reverse-transcription PCRfrom 4 populations of A mellifera, 4 sequences from 1 populationbelonged to the 2134D51 genotype (349 nucleotides, nt) and 12sequences from 3 populations belonged to the 2100D0 genotype (400nt) among the 16 determined sequences A total of 87 points ofmismatches were found by comparison with the most similarsequences in GenBank Seventeen single-nucleotide polymorphisms(SNP) were detected, and 6 SNP-patterns in the 2100D0 genotypeand 2 SNP-patterns in the 2134D51 genotype were identified based

on SNP positions In SNP-pattern 2, 10 SNPs were detected, but only

2 SNPs were found in SNP-pattern7 Meanwhile, one SNP-patternwas found from one RNA sample, multi SNP-patterns were detectedfrom other RNA-samples Large numbers of SNP variants indicatethat vast numbers of point-mutations on SBV have occurred sinceSBV outbreak in Korea and that SNPs may have been appearedindividually over time Thorough analysis of SNP variants will notonly define the local infection-route, but also the relationships

between SNP-pattern and SBV pathogenic abilities.

Israeli acute paralysis virus (IAPV) was demonstrated to involve inColony Collapse Disorder (CCD) of honey bees in USA However,because the virus exhibits a high level of genetic variation and someIAPV strains exhibit high degrees of homology with related viruses,the detection of IAPV in infected honey bees is relatively challengingwork To overcome these obstacles, a new molecular approach wasdeveloped, that relies on multiple detection sites within the IAPVgenome and on UR-qPCR The new system simultaneously targeted aRNA-dependent RNA polymerase gene (RdRp) and two capsid genes(VP3 and VP1) This multi-point PCR approach was highly efficient,with the ability to detect 100% of IAPV infections, and outperformedsingle-point PCR, which was only able to detect 86.96–95.6% of IAPVinfections Sequence analysis indicated that RdRp was more variablethan the two capsid genes, and the specificity of the proposed methodwas demonstrated by the detection of IAPV from samples co-infected

by IAPV and Kashmir bee virus (KBV) Both freezing-thawing RNAisolation and UR-qPCR could be performed in 27 min and 40 s.Therefore, this new application provides a useful tool for the rapididentification of IAPV in apiaries

European foulbrood (EFB) is a bacterial disease caused by

Melissococcus plutonius The larvae in infected colony are commonlykilled by the bacteria and emit a foul smell A quantitative analysisfor accurate diagnosis of the EFB pathogen is important to determinethe level of infections by which an effective treatment could beapplied The current detection methods, microscopic count, plate count,and molecular count relying on UR-qPCR were evaluated in thisstudy The result showed that the plate count and microscopic countwere laborious for the accurate quantification of M plutonius because

the bacteria tend to attach together like a chain The molecular countshow the minimum limit of 21 copies of M plutonius DNA to bedetected, and the relationship between the DNA copy and bacterianumber was established Furthermore, the PCR analysis showed theadvantage in infected larvae without symptom of EFB in apiary Theartificial infection of M plutonius to larvae result showed that thehigher survival and development rate of larvae increase in olderlarvae when all exposed to the same quantity of bacteria For theadvantages of sensitivity, rapidity, the UR-qPCR is expected to bethe reliable tool for accurate diagnosis of level of M plutonius

infection in honeybee

The microsporidian parasite Nosema ceranae is a global problem inhoneybee populations and is known to cause high winter mortality ofbee colonies This study developed the quantitative method thatincorporates ultra-rapid real-time PCR (UR-qPCR) for the rapidenumeration of N ceranae in infected adult bees UR-qPCR wasmore sensitive than microscopic enumeration for detecting two copies

of N ceranae DNA, and 1.91 × 102 spores per bee Meanwhile,microscopic detection showed a limit of detection was 1.91 × 104

purified spores/ml, and the stable detection level was ≥1.91 × 105

spores/ml The result of N ceranae calculation from the infectedhoneybees showed that the number of DNA copy was around 8.00fold higher than spore number, and the samples infected by N.ceranae with 104 to 105 copies DNA copies/bee was laborious to bedetected by microscopic method

Monoclonal antibody (mAb) was produced for immunologicalmethodology detection of Nosema ceranae Mouse was initiallyimmunized by 107 spores, and then 71 µg of total protein wasprepared for each boosting injection Hybridoma for specific mAb

production was produced after 11 weeks of the immunization period.The result of Western-blot using mAb showed that the mAb detectsthe protein of N ceranae around 17kDa, and Dot-blot assay had thepositive detection from 2 × 106 to 2 × 103 spores of N ceranae.Ascitic fluid was produced in peritoneal cavity of mice for a largeamount of antibody The result of ELISA showed the sensitivity of

N ceranae detection using ascitic fluid was much higher than thecell cultural medium in all dilution rate As a result, this antibodycould be used to produce cheap immunochromatographic strip forroutine inspection of N ceranae in apiary

Honey is the natural sweet syrup collected from flowers byhoneybee, and is widely used in human life for the appreciated tasteand health benefits, so that, expensive honey has been targeted to befaked from economic desire High-fructose corn syrup (HFCS) hasbeen prevalently used for the adulteration of honey In the presentstudy, a molecular method was developed for adulterated honeydetection by targeting on the specific gene of Maize Maize residualDNA in the honey was detected using genomic, mitochondria, andchloroplast specific primers These primers were designed tospecifically detect certain variety from maize variants The result ofquantitative detection showed that the honey that adulterated with 20,

40, 60, and 80 % of corn syrup can be detected based on thecomparison of quantity of genomic DNA in adulterated honey withnatural honey The commercial seven honey samples were all detectedwith the presence of maize DNA However, the quantity were alllower than that in the adulteration ratio This method was expected

to be a useful tool for rapid determination of the corn syrupadulterated honey

Monofloral honey is produced by the predominant nectar of a single

botanical species, and has its own unique properties, functional, ormedical properties, therefore it is targeted to be adulterated byincorrect labelling or mixing with other cheaper honey In this study,

a molecular authentication method for monofloral honey and theseason of honey production was developed The major nectar plants

Prunus sp., Robinia pseudoacacia, Castanea sp., Tetradium sp., and

Kalopanax sp were selected for identification of honey produced inApril, May, June, and July, respectively In twenty honey samplescollected in the four months, quantitative analysis from PCR showedthat the DNA of each plant was detected with highest amount inflowering season and was remained in the samples of the followingmonths Additionally, the plant composition existed in the honeysamples increased from May to July However, the DNA quantity ofeach plant species tended to decrease The confirmative result ofcommercial monofloral honey showed that only the cherry blossomhoney showed the majority of Prunus DNA Other monofloral honeyshad the mislabeling due to the lower quantity of the expected DNAthan others This molecular tool is expected to be useful to verify theseasonal honey and to verify the origin plant of monofloral honey

Part 1 Molecular characterization of honeybee pathogens

SBV was first recorded in 1913, but was not characterized until 1964(White, 1917; Bailey et al., 1964) It has been found worldwide, includingNorth America (van Engelsdorp et al., 2009), South America (Freiberg etal., 2012), Europe (Grabensteiner et al., 2001; Tentcheva et al., 2004),Australia (Anderson and Gibbs, 1988), South Africa (Grabensteiner et al.,2001), and Asia (Zhang et al., 2001; Choi et al., 2010; Ma et al., 2013;Nguyen and Le, 2013)

After many geographical variants of SBV have been detected, theseregional SBV variants were called as common name, such as cSBV(Chinese variant; Mingxiao et al., 2011), kSBV (SBV in Korea; Choi et al.,2010; Choe et al., 2012), vSBV (SBV in Vietnam; Nguyen and Le, 2013),iSBV (SBV in India; Kshirsagar et al., 1982; Rao et al., 2016), and tSBV(SBV in Thailand) In contrast, the first isolated SBV group, from westernhoneybee Apis mellifera, has been already worldwide detected from A.mellifera, was called simply as wSBV (western SBV; Lee et al., 2017).

2 Genotypes of Sacbrood virus

The mutation of SBV genome frequently occurred in the gene, locatedbetween VP1 and VP3 genes, encodes for a small capsid protein that wasnamed minor capsid protein (MiCP) This membrane protein wasdemonstrated to facilitate the infection of SBV (Procházková et al., 2018).Such mutation showed a similar trend in the SBV trains detected in eachregion It was considered to be the sign for SBV genotyping (Lee et al.,2017) Based on amino acid sequences of SBVs, Lee et al (2017) found acharacteristic deletion, which located on the MiCP gene Using position andnucleotides of this deletion, designated 2100D, five SBV genotypes wereproposed, such as 2134D51, 2119D39, 2119D30, 2100D0, and 2134D3 Thesegenotypes were also well matched to common names of SBV, such askSBV and vSBV, cSBV, iSBV, wSBV, and English SBV (eSBV),respectively

Some genotypes were recorded to have higher pathogenicity than anothersuch as the two genotypes, SBVD0 and SBVD51, were existed in Korea,but only SBVD51 was known to be the main factor that caused the

collapse of A cerana in Korea during the year 2010 (Lee et al., 2010; Choe

et al., 2012; Choi et al., 2010) Furthermore, co-infection of the two SBVgenotypes in one host, A mellifera, was recorded (Gong et al., 2016) Theorigin of these SBV genotypes and whether the variant in onegeographical region is able to infect honeybees in another region is aninteresting and unresolved issue Furthermore, the characteristics of eachgenotype are still unclear, and a tool for fast detection and accuratedifferentiation of these geographical SBV variants has not been developed

3 Detection of SBV

SBV can persistently exist in honeybee colony without clear symptoms

in adult bee, particularly when the virus accumulated in brains of infectedbees (Bailey and Fernando, 1972) Hence, besides the observation ofsymptoms in infected honeybees, the molecular detection by reversetranscription-PCR (RT-PCR) has been an accurate and rapid means ofvirus detection when the genomic nucleotide sequences of SBV have beendetermined (Grabensteiner et al., 2001) A PCR detection method for kSBVwas developed by Nguyen Thi et al (2009) Then SBV detection usingultra-rapid real-time PCR was developed by Yoo et al (2012) for detectionresult within 22 minutes including reverse transcription step This SBVdetection method has been greatly improved for rapid detection, forinstance, an ultra-rapid RT-PCR against kSBV that can detect the viruswithin 6 minutes 12 seconds (Min et al., 2016)

4 Purpose of this study

This study was conducted to develop a method for accurate and fast

determination of SBV genotypes using ultra-rapid real-time PCR Universal primer pair was designed to detect all SBV genotype, and followed by genotyping PCR using specific primer A method based on the quantitative nested PCR was also developed for calculation of SBV genotypes in infected honeybee samples The method was expected to be the most rapid method of genotype detection and quantification It plays an important role in establishing further research on the origin and infection patterns of each genotype by which a honey bee variant with resistance to SBV might be found.

II Materials and methods

1 Bacterial strain and plasmid vector for molecular

cloning

The Escherichia coli (E coli) strain DH5αF’, genotype F´/endA1 hsdR17(rK– mK+) glnV44 thi-1 recA1 gyrA (NalR) relA1 Δ(lacIZYA-argF)U169deoR (φ80dlacΔ(lacZ)M15), was widely used for cloning with highefficiency of transformation

Plasmid pBlueXcm (Figure 1) was used for cloning and sequencing, thisvector originated from pBluescript II KS (+) (Stratagene, U.S.) Therestriction of Xcm I on the multiple cloning site allows the creation ofthymine overhangs at the 3' that have complementary to adenine at the 3’end of PCR product using Taq DNA polymerase Therefore, the cloningcan be carried out by directly inserting PCR product into the vector thatwas cut by Xcm I enzyme The Ampicillin resistant gene (Ampr)) and β-galactosidase subunit (LacZ) was used for the screening of successfulcloning

Figure 1 Genetic map of pBlueXcm vector

Vector size is 3505 bp long. XcmI restriction site located on the multiple cloning site Amp r and LacZ represent the ampicillin resistant gene and β-galactosidase subunit, respectively.

2 Bacterial culture medium

The Luria-Bertani (LB) medium (Table 1) without ampicillin was used for the cultivation of bacteria that were used to produce the competent cell, and the LB medium with 50 µg/ml of ampicillin was used for selection of transformed-E.coli.

LB plate with 1% agar was used for cultivation of E.coli (Table 2) The plate with ampicillin supplement was used for screening of successful cloning colony.

To prepare the plate, the mixture of the compositions was autoclaved at 121 °C

for 20 min, and cooled down to 50-55 °C, then the ampicillin was added After well mixing the solution was poured into sterilized plates The plates were then packed and stored at 4 °C.

Table 1 Composition of Luria-Bertani liquid medium

Table 2 Composition of Luria-Bertani 1% agar media

3 SBV-infected honeybee samples

Ten honeybee samples, A mellifera, used for quantitative nested PCRwere collected in various regions in South Korea in which samples Yo1,Yo2, Yo3, Yo7 were collected in Yongin (2018); Su4, Su8, Su9, and Su10were from Suwon (2018); and the larvae samples, Ok5 and Ok6, werecollected in Okchon (2015) These samples were stored in –20 °C

4 Plasmid DNA isolation

The E.coli carrying plasmid pBlueXcm was cultivated at 37 °C for 16

hours in LB medium with ampicillin (50 µg/ml) The bacteria werecollected by centrifugation at 12,000 rpm for 30 s, 3 ml of the cultivatedsolution was used Plasmid was extracted from the collected bacteria using

Biotechnology, Korea) Pelleted bacterial cells were re-suspended in 150µlResuspension Buffer by pipetting, and 150µl Lysis Buffer was added andmix gently by inverting the tube 6 - 8 times, after that 350µl ofNeutralization Buffer was added and quickly mixing by inverting 12 - 20times, the solution was centrifuged for 2 min at 12,000rpm using atable-top microcentrifuge The supernatant was transferred to a SpinColumn without touching the pellet and centrifuging for 30 s at 12,000rpm The column was washed using 300µl Washing Buffer, andadditionally centrifuged for 1 min at 12,000 rpm to eliminate the residualWashing Buffer Spin Column was transferred to a new 1.5 mlmicrocentrifuge tube, and 50 µl Elution Buffer was added to the SpinColumn and centrifuging for 30 s at 12,000 rpm The DNA concentrationwas identified using a Biophotometer (Eppendorf, Germany)

5 Total RNA isolation and standard DNA construction

RNAiso Plus kit (Takara, Japan) was used for total RNA isolation fromhoneybee samples Two individuals of each sample were transferred into a

2 ml centrifuging tube that containing 1 ml of RNAiso Plus solution and

20 glass beads The bees were homogenized using a Magna lyser (Roche,Switzerland) The homogenate was incubated at room temperature for 5min before centrifuged at 12,000 ×G for 5 min at 4 °C The upper phase ofsolution was transferred to a new 1.5 ml tube, and 200 µl of Chloroformwas added and mixed well by vortexing After incubating at room

temperature for 5 min, the solution was centrifuged at 12,000 ×G for 15min at 4 °C The top layer of the supernatant was transferred to a new1.5 ml tube and mixed with 500 µl of isopropanol After 10 min incubating

at room temperature the solution was centrifuged at 12,000 ×G for 10 min,and remove the supernatant The pellet was finally washed using 1 ml of

75 % ethanol, after centrifuging at 10,000 ×G, the ethanol was eliminated,and pellet was kept at room temperature until dry The precipitated anddried RNA was dissolved in 100 μl of RNase-free water Concentration ofRNA was determined by using a Biophotometer (Eppendorf, Germany), anddiluted into concentration of 100 ng/μl that was used for SBV detection

6 Primer design

Two universal primer pairs were designed to detect all SBV strains:SBVD-F1/SBVD-R1 and SBVD-F2/SBVD-R2 (Table 3) A DNA fragment582-bp long was amplified from coding sequence (CDS) of genotype2100D0 by primer pair SBVD-F1/SBVD-R1 Size of amplicon in othergenotypes were 579bp (2134D3), 552bp (2119D30), 543bp (2119D39), and531bp long (2134D51) (Figure 2) This primer pair was used for SBVdetection from honeybee samples and for 2134D51 and 2100D0 standardDNA construction The second universal primer pair, SBVD-F2/SBVD-R2,was designed to combine with genotyping primers for SBV genotypesidentification (Figure 3)

Primers for specific genotype detection were designed based on the result

of nucleotide alignment Each genotyping primer was selected at theposition that contained the missing gap By this method the non-specificamplification can be avoided Five specific genotyping primers (Table 3)were designed to enable the accurate differentiation of 5 SBV genotypes

from infected honeybee samples.

Table 3 Primers used for SBV detection from honeybee samples andgenotyping PCR

Genotype

detection Primername Primer sequences(5’-3’)

Primer length (Mer)

Tm ( ° C) References2134D51 SBVD51-

F AGACCAAGAAGAGAATCAG 19 51.16 This study

Figure 2 The alignment of SBV genomes shows various numbers ofmissing nucleotide among the 5 genotypes from which the specificgenotyping primers were designed

The alignment shows 51, 39, 30, and 3 nucleotides were deleted in themissing gap of genotype 2134D51 (kSBV and vSBV), 2119D39 (cSBV),2119D30 (iSBV), 2134D3 (eSBV), respectively These deleting nucleotideswere identified based on the comparison with genotype 2100D0 (wSBV).Specific primer of each genotype was selected containing the deleting gap

in order to reduce the non-specific amplification Universal primer pairsSBVD-F1/SBVD-R1 and SBVD-F2/SBVD-R2 were designed to amplified

a fragment 582bp (1906-2487) and 288bp long (2065-2352), respectively, onCDS of 2100D0 genotype In the figure, only nucleotides at primerdesigning and deleting positions are shown This figure were created based

on the analysis of Lee et al (2017)

Figure 3 Schematic diagram shows primer pairs and amplicon size

of 2 μl of 10× reverse transcription buffer, 4 μl of 25 mM MgCl2, 2 μl of0.1 M DTT, 1 μl of RNaseOUTTM and 1 μl of SuperScript ® III reversetranscriptase was added After gently mixing the mixture was incubated at50°C for 50 min, followed by 85°C for 5 min

8 Standard DNA construction

The cDNA was directly used for PCR with the AccuPower® Taq PCRPreMix (Bioneer, Korea) and conventional PCR machine One- μl of cDNA,

1 μl of each primer (SBVD-F1/R1), and 17 μl of distilled water composed

a total 20 μl reaction mix PCR condition was 95°C (5 min), followed by 35cycles of 95 °C (30 s) - 53 °C (30 s) - 72 °C (30 s), and finally 72 °C (10min) PCR product was purified using the QIAquick PCR Purification Kit(QIAGEN, Germany) Purified DNA, 582 bp, of SBVD0 amplified fromsample Yo1 was inserted into plasmid pTOP TA V2 using TOPcloner™

TA core Kit (Enzynomics, South Korea), the recombinant plasmid wasdesignated as pSBVD0 The 531-bp long standard fragment of genotype2134D51 amplified honeybee sample (A cerana) collected in Okcheon, SouthKorea (2014) was inserted in the pBlueXcm vector by the TA cloningmethod using restriction enzyme XcmI and T4 ligase (New EnglandBiolabs, USA) The recombinant vector was designated as pSBVD51

Due to the lack of real infected samples for genotype 2119D30 (cSBV),2119D39 (iSBV), and 2134D3 (eSBV), the standard DNA of thesegenotypes were constructed based on the sequence information of thesegenotypes on NCBI A chemical synthesized oligo nucleotide (Table 4) thatcontains a part for detection of specific genotyping primer was used toplus another part derived from standard DNA of genotype 2100D0 or2134D51 For the construction of SBVD39 and SBVD30 standard fragments,the forward oligo fragments were paired with the universal primerSBVD-R1 for amplification of the first fragment on DNA template ofSBVD51, and the reverse oligo fragments were pair with primer SBVD-F1for the amplification of the second fragment on SBVD51 DNA template.Afterwards, the two fragments were joined together and inserted into

plasmid pBlueXcm that was cut by enzyme XcmI The construction ofSBVD3 fragment was carried out in the same method except the DNAtemplate was from SBVD0 standard DNA.

Table 4 Chemical synthesized oligo nucleotides used for standardDNA construction

9 Specific identification of genotyping DNA

Each genotyping primer pair was separately inspected on standard DNAs

of 5 genotypes to confirm the specific amplification Additionally, themixture consisting standard DNA of 5 genotypes (106 molecules of eachgenotype) was utilized for each genotyping primer pair to examine thespecific detection PCR conditions were: 95ºC (30s) and 35 cycles of 95ºC(3s) - 54ºC (3s) - 72ºC (3s) GENECHECKER™ Ultra-Rapid Real-timePCR (UR-qPCR) and reagent 2X One-Step RT-PCR Master Mix(Genesystem Co., Ltd., Korea) were used

10 Sensitivity of SBV detection

Sensitivity of each SBV genotype detection was assessed using

genotyping primers and recombinant DNAs Recombinant DNAs wereserially diluted from 108 to 100 copy/µl, then 2 µl of each concentration wasused for genotyping PCR A standard curve represents the relationshipbetween threshold cycle (Ct) and initial copy of DNA template wasestablished for each genotyping primer pair PCR condition was 95°C (30s),

50 cycles of 95 °C (3s)-54 °C (3s)-72 °C (3s)

Sensitivity of SBV genotyping was also inspected in nested PCR, SBVwas detected by universal primer pair SBVD-F1/R1, and PCR product was500× diluted and used for genotyping PCR Serial dilution of each standardDNA was used for the inspection

11 Quantitative detection of SBV genotypes

DNA copy of each SBV genotype was calculated in total RNA isolatedfrom infected honeybee in quantitative nested PCR based on the method ofTakahashi and Nakayama (2006) and Takahashi et al (2008) The RT-PCRconditions were: 5 min at 50 ºC for RT, followed by PCR at 95ºC (30s)and 35 cycles of 95 ºC (4s)-51 ºC (4s)-72 ºC (4s) PCR product was 500×diluted and used for nested PCR (genotyping PCR) at PCR condition of95ºC (30s), and followed by 35 cycles of 95 ºC (3s) - 54 ºC (3s) - 72 ºC(3s) GENECHECKER™ Ultra-Rapid Real-time PCR (UR-qPCR) andreagent 2X One-Step RT-PCR Master Mix (Genesystem Co., Ltd., Korea)were used

DNA copy of each genotype was calculated using the standardregression of each genotyping primer pair, then the initial DNA copy ofeach genotype in 100 ng total RNA was calculated by comparing to the

amplification of standard DNA using the formula bellow:

X:W = m:M ⸫ X = W×m/M

In the equation, X and m were the initial DNA copy of each genotype in 100 ng total RNA and initial number (10 3 copies) of standard DNA used in detection PCR, respectively; W and M were copy number of sample DNA and standard DNA that was calculated in nested PCR using genotyping specific primer.

12 Agarose gel electrophoresis

To confirm the accurate amplification of target DNA, electrophoresis wasconducted using 1.5% agarose After running the electrophoresis in 1×TAE buffer (Table 5) the gel was stained in Ethidium bromide solution (1µg/ml), and observing the band of DNA under UV light

Table 5 Composition of TAE buffer

Note: The buffer was prepared by 50×, and was then diluted to 1× for thefinal use

III Results and discussion

1 Standard DNAs for SBV genotyping

The standard DNAs of 5 genotypes were constructed, and used aspositive control for genotyping identification The position of these

TAE buffer Composition for 50× stock solution Amount