Development And Optimisation For Molecular-based Rapid Detection . - Core

DEVELOPMENT AND OPTIMISATION FOR MOLECULAR-BASED RAPID DETECTION OF SALMONELLA IN RAW DUCK MEATS AND MUNG BEAN SPROUTS ZHENG QIANWANG NATIONAL UNIVERSITY OF SINGAPORE 2015

DEVELOPMENT AND OPTIMISATION FOR MOLECULAR-BASED RAPID DETECTION OF SALMONELLA IN RAW DUCK MEATS AND MUNG BEAN SPROUTS ZHENG QIANWANG (B. Sc. Sun Yat-Sen University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2015

THESIS DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety, under the supervision of Dr Yuk Hyun-Gyun. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. The content of the thesis has been partly published in: 1. Zheng, Q., Bustandi, C., Yang, Y., Schneider, K.R., Yuk, H.G. 2013. Comparison of enrichment broths for the recovery of healthy and heatinjured Salmonella Typhimurium on raw duck wings. Journal of Food Protection 76:1963-1938. 2. Zheng, Q., Mika-Krajnik, M., Yang, Y., Xu, W., Yuk, H.G. 2014. Real-time PCR method combined with immunomagnetic separation for detecting healthy and heat-injured Salmonella Typhimurium on raw duck wings. Internationl Journal of Food Microbiology 186:6-13. 3. Zheng, Q., Mika-Krajnik, M., D’Souza, C., Yang, Y., Heo, D.J., Kim, S.K., Lee, S.C., Yuk, H.G. 2015. Growth of healthy and sanitizerinjured Salmonella cells on mung bean sprouts in different commercial enrichment broths, with an emphasis on failure of lactose broth for Salmonella enrichment. Food Microbiology 52: 159-168. Zheng Qianwang 6 August 2015 I

ACKNOWLEDGEMENTS First and foremost, I would like to express my special thanks of gratitude to my supervisor Dr. Yuk Hyun-Gyun. He is knowledgeable and creative, and therefore can always provide me with valuable information and enlighten me when my project is in difficulties. He also provides us with valuable opportunities to attend some international competitions and conferences. He is always patient to guide us on both experiment and writing skills. And he is also kind and willing to give us help on campus life. Next, I would like to express my sincere thanks towards Dr MikšKrajnik Marta, Miss Yang Yishan in our group, who provides me with precious advise in experiment and manuscript writing. I also thank my other dear labmates and previous honors student, Hazel, Minjeong, Vinayak, Xinyi, Wenqian, Craig, and Caroline for their help in experiment and for all the girls that makes my life in Singapore wonderful. I also want to express my gratitude towards other members in our programme that provided technical support when I was in need. I am thankful to the technical assistance and other help from the staff in Food Science and Technology Programme. I am also thankful to other professors in our programme that provides precious help and advise. Last but not the least, I would like the thank my dearest friends, Zhang Xi and Wang Jiaqi, for their unconditional help when I was really in need in Singapore. And I would like the extend my gratitude toward my family: my parents Zheng Jian and Xie Chuhong, my husband Lin Heng, my grandparents and my parents-in-law, who support me financially and spiritually throughout my four years in Singapore and my life. II

LIST OF PUBLICATIONS AND MANUSCRIPTS List of publications and manuscripts derived from this thesis 1. Zheng, Q., Bustandi, C., Yang, Y., Schneider, K.R., Yuk, H.G. 2013. Comparison of enrichment broths for the recovery of healthy and heatinjured Salmonella Typhimurium on raw duck wings. Journal of Food Protection. 76:1963-1938. 2. Zheng, Q., Mika-Krajnik, M., Yang, Y., Xu, W., Yuk, H.G. 2014. Real-time PCR method combined with immunomagnetic separation for detecting healthy and heat-injured Salmonella Typhimurium on raw duck wings. International Journal of Food Microbiology. 186:6-13. 3. Zheng, Q., Mika-Krajnik, M., D’Souza, C., Yang, Y., Heo, D.J., Kim, S.K., Lee, S.C., Yuk, H.G. 2015. Growth of healthy and sanitizerinjured Salmonella cells on mung bean sprouts in different commercial enrichment broths, with an emphasis on failure of lactose broth for Salmonella enrichment. Food Microbiology. 52: 159-168. 4. Zheng, Q., Mika-Krajnik, M., Yang, Y., Lee, S.M., Lee, S.C., Yuk, H.G. 2015. Evaluation of real-time PCR coupled with different pretreatments for the detection of healthy and sanitizer-injured Salmonella spp. on mung bean sprouts. International Journal of Food Microbiology. (Under revision). List of related publications and manuscripts 1. Zheng, Q., Mika-Krajnik, M., Yang, Y., Yuk, H.G. 2015. Improving the detection accuracy of BAX system on Salmonella Enteritidis on egg related products by broth optimization and PCR inhibitors removal. (Under preparation). III

TABLE OF CONTENTS Page THESIS DECLARATION . I ACKNOWLEDGEMENTS . II LIST OF PUBLICATIONS AND MANUSCRIPTS . III TABLE OF CONTENTS .IV SUMMARY . VII LIST OF TABLES .IX LIST OF FIGURES . XI LIST OF ABBREVIATIONS .XIII LIST OF SYMBOLS . XV CHAPTER 1 Introduction . 1 1.1 Research background. 1 1.2 Research objectives . 5 CHAPTER 2 Literature review . 8 2.1 Duck meat and mung bean sprouts . 8 2.2 Salmonella . 9 2.2.1 Features and taxonomy . 9 2.2.2 Salmonella as pathogens. 9 2.2.3 Salmonella outbreaks .11 2.2.4 Environmental factors affecting Salmonella survival .14 2.3 Conventional culture method for Salmonella detection. 16 2.4 Rapid method for Salmonella detection . 16 2.4.1 Enrichment step .18 2.4.2 Sample pre-treatments .22 2.3.3 Downstream detection methods .28 CHAPTER 3 Comparison of enrichment broths for the recovery of healthy and heat-injured Salmonella Typhimurium on raw duck wings. 36 3.1 Introduction. 36 3.2 Materials and methods. 38 3.2.1 Bacterial cultures .38 3.2.2. Inoculation on raw duck wings .38 3.2.3 Growth kinetics of S. Typhimurium in enrichment broths.39 3.2.4 Recovery of heat-injured and healthy cells at low inoculum levels.40 3.2.5 Enumeration.42 3.2.6 Data analysis .42 3.3 Results . 42 3.3.1 Growth parameters of S. Typhimurium in various enrichment broths.42 3.3.2 Enrichment of healthy Salmonella cells at low inoculum levels in selected broths .44 3.3.3 Enrichment of heat-injured Salmonella cells at low inoculum levels in selected broths.46 3.4 Discussion . 49 IV

3.5 Conclusion. 51 CHAPTER 4 Real-time PCR method combined with immunomagnetic separation for detecting healthy and heat-injured Salmonella Typhimurium on raw duck wings . 52 4.1 Introduction. 52 4.2 Materials and methods. 53 4.2.1 Preparation of healthy and heat-injured Salmonella cells .53 4.2.2 Inoculation of Salmonella cells on raw duck wings .54 4.2.3 Optimisation of IMS .54 4.2.4 Optimisation of real-time PCR.55 4.2.5 Standard ISO method and real-time PCR for Salmonella detection .58 4.2.6 Validation.60 4.2.7 Statistical analysis.60 4.3 Results . 61 4.3.1 Optimisation of IMS .61 4.3.2 Optimisation of real-time PCR.62 4.3.3 Comparison of the optimised real-time PCR with IMS and conventional real-time PCR .67 4.3.4 Validation.67 4.4 Discussion . 70 4.5 Conclusion. 74 CHAPTER 5 Growth of healthy and sanitiser-injured Salmonella cells on mung bean sprouts in different commercial enrichment broth . 76 5.1 Introduction. 76 5.2 Materials and methods. 78 5.2.1 Bacterial cultures .78 5.2.2 Preparation of healthy and sanitiser-injured Salmonella cells .78 5.2.3 Inoculation.79 5.2.4 Growth kinetics of S. Typhimurium in different enrichment broths .80 5.2.5 Growth of healthy and sanitizer-injured cells at low inoculum levels .80 5.2.6 Enumeration.80 5.2.7 Investigation of LB inhibition mechanism of Salmonella on mung bean sprouts.81 5.2.8 Data analysis .84 5.3 Results . 84 5.3.1 Growth parameters of S. Typhimurium in enrichment broths .84 5.3.2 Enrichment of healthy and 90% sanitiser-injured cells at low inoculum levels in AD, OB and BPW .87 5.3.3 The inhibition mechanism of LB during Salmonella enrichment .90 5.4 Discussion . 96 5.5 Conclusion. 102 CHAPTER 6 Evaluation of real-time PCR coupled with different pretreatments for the detection of healthy and sanitiser-injured Salmonella spp. on mung bean sprouts. 103 6.1 Introduction. 103 6.2 Materials and methods. 104 6.2.1 Bacterial cultures . 104 6.2.2 Preparation of healthy and sanitiser-injured Salmonella cells . 105 6.2.3 Inoculation. 105 6.2.4 IMS . 105 6.2.5 Real-time PCR . 106 6.2.6 Detection probability and LOD . 106 V

6.2.7 Comparison of real-time PCR with standard ISO method for Salmonella detection . 107 6.2.8 Validation. 107 6.2.9 Statistical analysis. 108 6.3 Results . 108 6.3.1 Optimisation of IMS . 108 6.3.2 Detection probability and LOD of PCR methods with different pretreatments . 112 6.3.3 Comparison of real-time PCR coupled with different pre-treatments with ISO method . 114 6.3.4 Validation. 117 6.4 Discussion . 121 6.5 Conclusion. 125 CHAPTER 7 General conclusions and future work . 126 7.1 General conclusions . 126 7.2 Future work . 128 BIBLIOGRAPHY . 130 VI

SUMMARY Salmonella is a leading cause of foodborne illnesses worldwide and poses a great potential threat to public health. To reduce salmonellosis, national regulatory bodies and food companies have used reliable and highly sensitive standard culture methods to monitor the presence of Salmonella in food products. However, the standard culture method is labour-intensive and time-consuming, usually taking up to 5 – 7 days to complete. Therefore, to obtain immediate information on Salmonella contamination, it is necessary to develop and implement a rapid detection method for Salmonella in foods. Raw duck meats and mung bean sprouts, which are widely consumed in Asian countries, were selected as model foods in this study. Three steps, including enrichment, sample pre-treatment, and real-time PCR detection, were optimised to develop a new diagnostic protocol for the rapid detection of Salmonella in duck meats and mung bean sprouts. Nine commercially available enrichment broths were evaluated for their effectiveness on the regrowth and recovery of healthy and injured Salmonella in the food samples. Immunomagnetic separation (IMS) and centrifugation as sample pre-treatment methods were used to concentrate and isolate bacteria from different food matrices, and their performances were evaluated. The condition of real-time PCR was optimised by comparing the performance of different primer sets. The effectiveness of the newly developed real-time PCR based detection method was evaluated and validated using both artificially inoculated and naturally contaminated food samples. Results showed that ONE broth-Salmonella (OB) and buffered peptone water (BPW) were suitable broths to enrich healthy and injured Salmonella in VII

duck meat and sprouts, respectively. However, lactose broth (LB) was unable to recover Salmonella to detectable levels in either duck or sprout samples, causing possible false negative results in Salmonella detection. IMS helped decrease the detection limit of PCR for duck meats, while comparable performance was observed in IMS and centrifugation for bean sprouts. Using Sal primer allowed lower detection limit and higher PCR efficiency than the invA and ttr primers. All inoculated samples were successfully detected by the newly developed real-time PCR based method. The diagnostic accuracy of real-time PCR coupled with IMS or centrifugation for duck meat and sprouts was shown to be 98.3% in the validation study. These results indicate that with appropriate enrichment and sample pretreatment steps, this newly developed and optimised real-time PCR based procedure could shorten the whole detection time to 16-h, compared with the 5-7 days detection period of standard culture methods. Thus, this new protocol might contribute to improve a monitoring/surveillance program and eventually to minimize the risk of salmonellosis. VIII