Title Page

Contents

ABSTRACT 9

Ⅰ. Introduction 10

Ⅱ. Materials and Methods 23

1. Ethical states 23

2. Cell line and viruses 23

3. in vitro infection 23

4. Clinical information about patient with CHIKV 24

5. RNA extraction and cDNA synthesis 26

6. Real Time quantitative Polymerase Chain Reaction (RT-qPCR) 29

7. Design of primers and probes for SARS-CoV-2 29

8. Sensitivity and specificity of SARS-CoV-2 primers by RT-qPCR 32

9. One-step multiplex RT-qPCR 32

10. Design of multiplex PCR primers for Chikungunya virus 32

11. Multiplex PCR for amplification of CHIKV 34

12. Amplicon-based MinION sequencing 38

13. NGS data analysis 38

14. Phylogenetic analysis 39

15. Prediction the zoonotic potential of the chikungunya virus 39

16. Statistical analysis 41

Ⅲ. Results 42

1. Comparative Evaluation of RNA Extraction Kits for SARS-CoV-2 42

2. Detection limit of each primer using Quantstudio3 and Franklin 45

3. Specificity of each primer using Quantstudio3 and Franklin 49

4. One-step multiplex RT-qPCR for SARS-CoV-2 using Quantstudio3 and Franklin 52

5. Comparative Analysis of SARS-CoV-2 Multiplex RT-qPCR Performance 57

6. Design of a multiplex PCR primer pool for chikungunya virus 60

7. Multiplex PCR-based Next-generation Sequencing for chikungunya virus from the Korean patient 63

8. Genotyping of chikungunya virus extracted from Korean patient 66

9. Predicting the zoonotic potential of the chikungunya virus 69

Ⅳ. Discussion 72

Ⅴ. References 76

국문초록 84

Table 1. Laboratory diagnosis of samples from patients with Chikungunya fever 25

Table 2. Primers and probe sequences used for the detection of SARS-CoV-2 31

Table 3. Primers sequences used for the multiplex PCR targeted chikungunya virus 35

Table 4. Characteristics of genomic sequences of chikungunya virus used in this study 40

Table 5. Regression Equations for SARS-CoV-2 Variant Detection in Two Thermocyclers 48

Table 6. Comparison of NGS result between Day 2 and Day 6 64

Table 7. Comparison of reference mapping result between Day 2 and Day 6 65

Figure 1. Zoonotic transmission routes 11

Figure 2. Point-of-care testing formats 13

Figure 3. Genome structure of Severe Acute Respiratory Coronavirus-2 15

Figure 4. Point-of-care testing using Biomeme Franklin™ three9 Real-Time PCR Thermocycler 17

Figure 5. Distribution of different lineages of the Chikungunya virus 19

Figure 6. MinION device and workflow of amplicon-based nanopore sequencing 21

Figure 7. Four commercial RNA extraction kits used in this study 28

Figure 8. SARS-CoV-2 spike gene specific primer pairs and probe set 30

Figure 9. Schematic diagram of multiplex PCR primer design for CHIKV. 33

Figure 10. Comparative Evaluation of RNA Extraction Kits for SARS-CoV-2. 44

Figure 11. Comparison of the detection limit of SARS-CoV-2 using a lab-scale qPCR machine and a portable qPCR machine. 47

Figure 12. Specificity Testing of SARS-CoV-2 Primers against Multiple Viral Pathogens. 51

Figure 13. Comparative Analysis for one-step multiplex real-time quantitative PCR using lab-scale and portable qPCR instruments. 54

Figure 14. Comparative analysis of one-step multiplex real-time quantitative PCR on lab-scale instrument and portable qPCR device based on results from in vitro samples. 56

Figure 15. Comparative analysis of qPCR instruments in singleplex and multiplex conditions for clinical nasopharyngeal specimens. 59

Figure 16. Multiplex PCR for Chikungunya virus from Korean patient 62

Figure 17. Phylogenetic analysis of CHIKV from Korean patient, and reference strains. 68

Figure 18. Prediction of zoonotic potential for chikungunya viruses 71