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

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

(2019)

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

- i -

Contents

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

- ii -

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

- iii -

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

- iv -

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

- v -

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

- vi -

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

- vii -

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

- viii -

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

- ix -

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

- x -

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

DNAs ····························································································50

<Table 14> Sequence of standard DNAs used as positive controls for

IAPV detection ···········································································50

<Table 15> Efficiency of single- and multi-point PCR for the

- xi -

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

- xii -

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 which

the 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 honeybee

samples ·························································································22

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

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

RNA-samples ··············································································34

<Figure 9> Alignment of 12 sequences from RNA-samples 1, 2, and

3 belonging to 2100D0 ······························································36

<Figure 10> SNP variants based on 400-bp sequences belonging to

genotype 2100D0 ······································································37

<Figure 11> Alignment of 4 sequences from RNA-samples 4

belonging to 2134D51 ······························································40

<Figure 12> SNP variants based on 349 nt sequences belonging to