blood sample 35 LIST OF List of tables Page Table 3.1: The information of PCR primers 16 Table 4.1: PCR and LAMP result of A. baumannii strains 20 Table 4.2: PCR and LAMP result of P. aeruginosa strains 27 Table 4.3: PCR and LAMP result of K. pneumoniae strains 32 LIST OF APPENDIX List of appendix page Appendix 1: Bacterial strains used for optimization 50 Appendix 2: Chemicals and reagents 53 Appendix 3: In silico PCR amplification of gltA F3, B3 from A. baumannii for LAMP primer 56 Appendix 4: In silico PCR amplification of gltA FIP from A. baumannii for LAMP primer 57 Appendix 5: In silico PCR amplification of gltA BIP from A. baumannii for LAMP primer 58 Appendix 6: In silico PCR amplification of ABC transporter permease F3, B3 from K. pneumoniae for LAMP primer 59 Appendix 7: In silico PCR amplification of ABC transporter permease FIP from K. pneumoniae for LAMP primer 60 Appendix 8: In silico PCR amplification of ABC transporter permease BIP from K. pneumonia for LAMP primer 61
Appendix 9: In silico PCR amplification of rimM F3, B3 from P. aeruginosa for LAMP primer 62 Appendix 10: In silico PCR amplification of rimM FIP from P. aeruginosa for LAMP primer 63 Appendix 11: In silico PCR amplification of rimM BIP from P. aeruginosa for LAMP primer 64 Appendix 12: Presentation and publication 65 ABBREVIATION AND SYMBOLS List of abbreviations and symbols
2.3 Pseudomonas aeruginosa Pseudomonas aeruginosa (P. aeruginosa) is a rod-shaped Gram-negative obligatory aerobic bacterium, belongs to the family of Pseudomonadaceae (Schwartz et al., 2006). It is one of the top opportunistic pathogens which are capable of causing NI when the host's resistance is low especially when there is a burnt-wound in the patient’s body (Stover et al., 2000) and is associated with cystic fibrosis (Filho et al., 1999). A lot of serious infections like pneumonia, otitis, endocarditis, septicemia and keratitis are caused by P. aeruginosa (Lavenir et al., 2007). Most P. aeruginosa are multiple drug-resistant and it exists widely in nature. P. aeruginosa strains also have developed resistance against antibiotics such as fluoroquinolones and even disinfectant (Schwartz et al., 2006). 2.4 Acinetobacter baumannii Acinetobacter baumannii (A. baumannii) is a Gram-negative bacterium, lives freely in water, soil and human skin, especially hands (Rungruanghiranya et al., 2005). The simplicity of its growth requirements and its high tolerance of environmental conditions results in more outbreaks caused by this pathogen (Chang et al., 2009). In Malaysia, the prevalence rate of nosocomial infection was 13.9% (Hughes et al., 2005). It can cause pneumonia, urinary tract infection, otitis media, catheter-related infection, central nervous system infection, peritonitis, and primary bloodstream infection (Levin et al., 1999). The main pathway of the infection is through the hospital equipment, especially the equipment which has direct contact with blood. The resistance of A. baumannii in fluoroquinolones, aminoglycosides and broad-spectrum b-lactams has been reported (Koeleman et al., 2001) and from the surveillance carried out from 2004
Fig 4.5: The detection limit of LAMP (a) and PCR (b) using A. baumannii spiked blood sample. 1 = 5.5×106 CFU/ml; 2 = 5.5×105 CFU/ml; 3 = 5.5×104 CFU/ml. Amplified product is seen for 1-2. 4.2 P. aeruginosa 4.2.1 Optimized LAMP Five temperatures were tested to determine the optimum temperature. These included 60℃, 62℃, 63℃, 64℃and 65℃ (Fig 4.6). Figure 4.6 shows that at 65℃ the reaction had the shortest amplification time. The primer for P. aeruginosa was different from the primers for A. baumannii and K. pneumoniae. This set of primer was combined with loop primers, so the amplification time would be much shorter. Fig 4.7 shows the difference between the amplification with and without loop primer. The whole reaction was run for 60 min at 65℃. With the inclusion of loop primers, the amplification of P. aeruginosa began at 25 min, loop primer made a 20 min saving of the reaction. Compared to the reaction of A. baumannii and K. pneumoniae, this reaction was faster. This shows that the ability of loop primer was to shorten the detection time. Fig 4.6: Comparison of LAMP results for P. aeruginosa PS19 at different temperatures Fig 4.7: Comparison of LAMP results with or without loop primer on PS19
49 K. pneumoniae K09-24 50 K. pneumoniae K09-25 51 K. pneumoniae K10-03 52 K. pneumoniae K10-04 53 K. pneumoniae K10-05 54 K. pneumoniae K11-01 55 K. pneumoniae K11-02 56 K. pneumoniae K11-03 57 K. pneumoniae K11-04 58 K. pneumoniae K11-05 59 K. pneumoniae K11-06 60 K. pneumoniae K11-09 61 P. aeruginosa PS2 62 P. aeruginosa PS16 63 P. aeruginosa PS19 64 P. aeruginosa PS20 65 P. aeruginosa PS23 66 P. aeruginosa PS67 67 P. aeruginosa PS98 68 P. aeruginosa PS100 69 P. aeruginosa PS102 70 P. aeruginosa PS103 71 P. aeruginosa PS105 72 P. aeruginosa PS108 73 P. aeruginosa 74 P. aeruginosa PS239 75 P. aeruginosa PS339 76 P. aeruginosa PS341 77 P. aeruginosa PS362 78 P. aeruginosa R01 79 P. aeruginosa R02 80 P. aeruginosa R04 81 P. aeruginosa 1182 82 P. aeruginosa 1186 83 P. aeruginosa 1260 84 P. aeruginosa 1288 85 P. aeruginosa B14141 86 P. aeruginosa B14143 87 P. aeruginosa B14128 88 P. aeruginosa B14262 89 P. aeruginosa B14349 90 P. aeruginosa BF2087 91 E. coli P49 92 E. coli P41 93 E. coli BS4 94 Vibrio VPD21 95 Vibrio VPD22 96 Vibrio 97 Vibrio VPD27 98 shigella TH32/98