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DEVELOPMENT OF RAPID LAMP ASSAYS FOR THE

DETECTION OF POTENTIAL NOSOCOMIAL PATHOGENS

DONG HONG

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FACULTY OF SCIENCE

UNIVERSITY OF MALAYA

KUALA LUMPUR

2013

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DEVELOPMENT OF RAPID LAMP ASSAYS FOR THE DETECTION OF POTENTIAL NOSOCOMIAL PATHOGENS

DONG HONG

DISSERTATION SUBMITTED IN FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF MASTER OF

BIOTECHNOLOGY

INSTITUTE OF BIOLOGICAL SCIENCES

FACULTY OF SCIENCE

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UNIVERSITY OF MALAYA

KUALA LUMPUR

2013

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UNIVERSITI MALAYA

ORIGINAL LITERARY WORK DECLARATION

Name of Candidate: Dong Hong (I.C/Passport No: G45811942 ) Registration/Matric No: SGF110009

Name of Degree: Master of Biotechnology

Title of Project Paper/Research Report/Dissertation/Thesis (“this Work”):

Development of rapid LAMP assays for the detection of potential nosocomial pathogens

Field of Study: Microbial Biotechnology I do solemnly and sincerely declare that:

(1) I am the sole author/writer of this Work;

(2) This Work is original;

(3) Any use of any work in which copyright exists was done by way of fair dealing and for permitted purposes and any excerpt or extract from, or reference to or reproduction of any copyright work has been disclosed expressly and sufficiently and the title of the Work and its authorship have been acknowledged in this Work;

(4) I do not have any actual knowledge nor do I ought reasonably to know that the making of this work constitutes an infringement of any copyright work;

(5) I hereby assign all and every rights in the copyright to this Work to the University of Malaya (“UM”), who henceforth shall be owner of the copyright in this Work and that any reproduction or use in any form or by any means whatsoever is prohibited without the written consent of UM having been first had and obtained;

(6) I am fully aware that if in the course of making this Work I have infringed any copyright whether intentionally or otherwise, I may be subjected to legal action or any other action as may be determined by UM.

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Candidate’s Signature Date

Subscribed and solemnly declared before, Witness’s Signature Date

Name:

Designation

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ABSTRACT

Nosocomial pathogens are organisms that cause diseases in a patient during his/her stay in a hospital or health care center. These pathogens can spread easily in the hospital and cause an outbreak because of the low immune system of the hospitalized patients.

Even so, they are resistant to most of the antibiotics. Rapid detection of these pathogens would be useful to trace the source of an outbreak. In this study, a new detection method, the loop-mediated isothermal amplification (LAMP) was developed for the rapid detection of three nosocomial pathogens which are highly multidrug resistant.

They are Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae.

Acinetobacter baumannii is a Gram-negative bacterium which can cause serious infection. It can cause wound infection, pneumonia, urinary tract infection, and etc. The LAMP primers for Acinetobacter baumannii were based on gltA gene and amplified in 62℃ for 90 min. The assay was evaluated on 50 bacterial strains, including 30 Acinetobacter baumannii and 20 non-Acinetobacter baumannii. All the positive strains were correctly identified, while the negatives were true negative. The sensitivity of LAMP was 5.5×104 CFU/ml, and it was 10-fold more sensitive than the normal PCR method (5.5×105 CFU/ml). The sensitivity of both LAMP and PCR were the same at 5.5×105 CFU/ml in spiked blood samples.

Pseudomonas aeruginosa causes pneumonia, otitis, endocarditis, septicemia and keratitis. The LAMP primers for P. aeruginosa were based on 16S rRNA-processing

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also evaluated on 50 strains (30 P. aeruginosa, 20 non-P. aeruginosa). The detection limit of this assay on bacterial culture was 3.6×104 CFU/ml, and it was 1000-fold more sensitive than PCR (3.6×107 CFU/ml). For spiked blood samples, the detection limit for LAMP was 7.7×104 CFU/ml which was 1000 fold higher than PCR (7.7×107 CFU/ml).

Klebsiella pneumoniae is a bacterial pathogen which causes pneumonia, bacteremia and meningitis. The LAMP primers were based on ABC transport permease gene and amplified at 65℃ for 90 min. The assay was evaluated on 50 bacterial strains, including 30 Klebsiella pneumoniae and 20 non-Klebsiella pneumoniae. There was no false positive or false negative result. The detection limit of LAMP was 7×103 CFU/ml, which was the same with normal PCR method. For spiked blood samples, the detection limit of both LAMP and PCR was the same at 1.4×104 CFU/ml.

Overall, LAMP is a rapid, effective and efficient assay which would contribute to the efficient detection of nosocomial pathogens.

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ABSTRAK

Patogen nosocomial adalah organisma yang menyebabkan penyakit dalam pesakit semasa beliau di hospital atau pusat penjagaan kesihatan. Patogen ini boleh merebak dengan mudah di hospital dan menyebabkan wabak kerana sistem imun yang rendah pada pesakit ke hospital. Tambahan, pathogen bakteria adalah menentang terhadap banyak antibiotik. Dalam kajian ini, kaedah pengesanan baru telah dioptimumkan untuk tiga patogen bakteria, iaitu, Acinetobacter baumannii, Pseudomonas aeruginosa dan Klebsiella pneumoniae. Kaedah ini dipanggil ‘loop-mediated isothermal amplification’

(LAMP) dan ia boleh mengesan jangkitan dalam fasa awal.

Acinetobacter baumannii adalah bakteria Gram-negatif yang boleh menyebabkan jangkitan yang serius. Ia boleh menyebabkan jangkitan luka, pneumonia, jangkitan saluran kencing, dan lain-lain. LAMP untuk Acinetobacter baumannii adalah berdasarkan gen gltA dan bertindak balas dalam 62℃ selama 90 min. Ujian ini dinilai dengan 50 strain bakteria, termasuk 30 Acinetobacter dan 20 bukan Acinetobacter.

Semua strain Acinetobacter dibukti positif manakala strain bukan Acinetobacter adalah negative. Had pengesanan adalah 5.5×104 CFU/ml, dan ia adalah 10 kali ganda lebih sensitif daripada kaedah PCR normal (5.5×105 CFU/ml). Sensitiviti kedua-dua LAMP dan PCR adalah sama sebanyak 5.5×105 CFU/ml bila diuji dengan sampel darah yang dicemari dengan strain AC090215.

Pseudomonas aeruginosa adalah bakteria yang boleh menyebabkan pneumonia, otitis, endokarditis, septisemia dan keratitis. The primer LAMP untuk P. aeruginosa

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65℃ selama 60 minit. Specificity primers untuk P. aeruginosa juga dinilai dengan 50 strain bakteria (30 P. aeruginosa, 20 bukan P. aeruginosa). Had pengesanan adalah 3.6×104 CFU/ml untuk LAMP, dan ia adalah 1000 kali ganda sensitif daripada PCR (3.6×107 CFU/ml). Had pengesanan adalah 7.7×104 CFU/ml dan kepekaan assay LAMP adalah lebih tinggi daripada PCR (1000-kali ganda) bila diuji dengan sampel darah yang dicemari dengan strain PS19.

Klebsiella pneumoniae adalah bakteria patogen yang menyebabkan pneumonia, bacteremia dan meningitis. Primer untuk ujian LAMP adalah berdasarkan gen tonB gene dan tindakbalas boleh diselesai pada suhu 65℃ dalam 90 min. Ujian ini dinilai dengan 50 strain termasuk 30 Klebsiella pneumoniae dan 20 strain bukan Klebsiella spp. Tiada positif palsu atau negative palsu dikesan. Had pengesanan adalah 7×103 CFU/ml, dan sama dengan kaedah PCR biasa. Kepekaan LAMP dan PCR adalah sama (1.4×104 CFU/ml) bila ujian dijalankan dengan sampel yang dicemari dengan strain K10-04.

Secara keseluruhan, LAMP adalah ujian yang cepat, cekap dan berkesan yang akan menyumbang kepada pengesanan jangkitan nosocomial

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ACKNOWLEDGEMENT

I would like to express the deepest appreciation to my supervisor Professor Dr.

Thong Kwai Lin for her patience guidance. And also thanks to all the lab mates in Laboratory of Biomedical Science and Molecular Microbiology.

I would like to express my special thanks to my parents for the long time's care and love. Without their support and help, this project would never be finished.

Thanks to University of Malaya for providing the PPP grants (P0024/2012A) for the research materials and reagents.

At last, thanks to all the people who have ever helped me during my research.

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TABLE OF CONTENT

Page

ABSTRACT...i

ABSTRAK...iii

ACKNOWLEDGEMENT...v

TABLE OF CONTENT...vi

LIST OF FIGURES...ix

LIST OF TABLES...x

LIST OF APPENDIX...xi

ABBREVIATIONS AND SYMBOLS...xiii

CHAPTER 1: INTROUCTION...1

1.1 General introduction...1

1.2 Objectives...3

CHAPETER 2: LITERATURE REVIEW...4

2.1 Definition of nosocomial infection...4

2.2 Klebsiella pneumoniae...5

2.3 Pseudomonas aeruginosa...6

2.4 Acinetobacter baumannii...6

2.5 Conventional detection methods for nosocomial pathogens...7

2.6 Loop-mediated isothermal amplification (LAMP) method...8

CHAPETER 3: METHODOLOGY...12

3.1 Materials...12

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3.1.1 Bacterial strains...12

3.1.2 Chemicals and reagents...12

3.2 Methods...12

3.2.1 DNA template...12

3.2.2 Primer design...13

3.2.3 LAMP reaction...13

3.2.4 Data analysis...14

3.2.5 PCR...15

3.2.6 Evaluation of sensitivity of LAMP assay in culture and blood samples17 CHAPTER 4: RESULTS...18

4.1 Primer designed for LAMP...18

4.2 Acinetobacter baumannii...18

4.2.1 Optimized LAMP assay...18

4.1.2 Sensitivity and specificity of the method...19

4.1.3Evaluation of LAMP on spiked blood sample...24

4.2 P. aeruginosa...25

4.2.1 Optimized LAMP assay...25

4.2.2 Sensitivity and specificity of the method...26

4.2.3Evaluation of LAMP on Spiked blood...29

4.3 K. pneumoniae...31

4.3.1 Optimized LAMP assay...31

4.3.2 Sensitivity and specificity of the method...31

4.3.3Evaluation of LAMP on spiked blood...34

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CHAPTER 6: CONCLUSION...41

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LIST OF FIGURES

List of figures Page Figure 2.1: Principle of LAMP 10

Figure 4.1: Comparison of LAMP results at different temperature of A. baumannii 19 Figure 4.2: Detection of the LAMP products using SYBR Green Ι 22

Figure 4.3: Visualization of LAMP products by agarose gel electrophoresis 22 Figure 4.4: Sensitivity of the LAMP assay (a) and PCR (b) for A. baumannii

AC 090 213 23 Figure 4.5: The detection limit of LAMP (a) and PCR (b) using A. baumannii spiked

blood sample 24

Figure 4.6: Comparison of LAMP results of P. aeruginosa PS19 at different

temperature 25

Figure 4.7: Comparison of LAMP results with or without loop primer on PS19 26 Figure 4.8: Sensitivity of the LAMP assay (a) and PCR (b) for P. aeruginosa PS19

29 Figure 4.9: The detection limit of LAMP (a) and PCR (b) using P. aeruginosa spiked

blood sample 30

Figure 4.10: Optimized temperature (65℃) of K. pneumoniae LAMP detection 31 Figure 4.11: Sensitivity of the LAMP assay (a) and PCR (b) for K. pneumoniae K10-04

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Figure 4.12: The detection limit of LAMP (a) and PCR (b) using K. pneumoniae spiked blood sample 35

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LIST OF TABLE

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

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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

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Appendix 12: Presentation and publication 65

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ABBREVIATION AND SYMBOLS

List of abbreviations and symbols

-Polymerase Chain Reaction -Deoxyribonucleic acid -Ribonucleic acid

-National center for Biotechnology Information -Basic Local Alignment Search Tools

-Luria Bertani -Tris Borate EDTA -Magnesium chloride

-deoxyribonucleotide triphosphates (dATP, dTTP, dCTP and dGTP) -Acinetobacter baumannii

-Escherichia coli

-Pseudomonas aeruginosa -Klebsiella pneumoniae -Salmonella Typhi

-Double distill/Deionized water -Ultraviolet

-Et alia (and other) -seconds

-minutes PCR

DNA RNA NCBI BLAST LB TBE MgCl2 dNTPs A. baumannii E. coli

P. aeruginosa K. pneumoniae S. Typhi ddH2O UV et al s

min

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℃ µM µL mL mM

-degree celsius -micromolar -microliter -milliliter -millimolar

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CHAPTER 1: INTRODUCTION

1.1 General introduction

Nosocomial infection (NI) is also known as hospital-acquired infection. It poses a significant problem worldwide. In the USA, there are roughly 1.7 million hospital- associated infections from all types of microorganisms, and that cause or contribute to 99,000 deaths each year. In Europe, Gram-negative infections are estimated to two-third of the 25,000 deaths each year (Pollack, 2010). The rapid spread of nosocomial pathogens has eventually increased the difficulty in treatment due to delayed detection and diagnosis. The difficult in treatment of nosocomial infection is also because of the multidrug resistance of the pathogen.

Usually, the detection and identification of nosocomial bacterial pathogen is performed by culture methods. A selective medium will be used for the identification, like the CHROMagar. However, the conventional culture methods are time consuming, usually need more than one day to detect up to genus level, and longer time is needed for species level identification. Because of the time consuming limitation of culture detection method, molecular method like Polymerase Chain Reaction (PCR) which is faster than conventional culture methods were introduced in medical diagnostics.

However, PCR also has some disadvantages. It needs additional steps like to run agarose gel electrophoresis continues with visualization of the products. So, it will take around 4 hours to finish the whole detection. The delay in detection may provide opportunity for pathogens to spread in the hospital. Therefore, it is essential to develop a more rapid and easier approach for the detection of nosocomial pathogens.

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In this study, a relatively new approach based on the loop-mediated isothermal amplification (LAMP) method is adopted, optimized and evaluated to determine its usefulness in detecting clinically important nosocomial bacterial pathogens. LAMP is one of the isothermal nucleic acid amplification methods and has received a lot of attention during the last decade because of its simplicity. It is an auto-cycling DNA synthesis which performed by four LAMP primers and DNA polymerase. Since the invention of LAMP method, it has already been developed for a lot of pathogens, like Mycobacterium (Iwamoto et al., 2003), Plasmodium falciparum (Poon et al., 2006), Shigella, Escherichia coli (Song et al., 2006), Streptococcus pneumoniae (Seki et al., 2005), Staphylococcus aureus (Lim et al., 2013) and many other bacterial pathogens.

With the help of Loopamp EXIA machine, the detection time can be shortened to 1.5 hours, or even 1 hour. This method can detect the bacteria in a really short time with less equipment and steps.

This thesis will focus on the development of LAMP assay for detection of three main nosocomial pathogens-A. baumannii, P. aeruginosa and K. pneumoniae, and the specificity and sensitivity were tested on bacterial cultures and spiked blood samples.

This LAMP assays were compared with the conventional PCR.

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1.2 Objectives

Overall, the goal of the study was to develop loop-mediated isothermal amplification (LAMP) assays for detection of Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa.

Specifically, the objectives were:

i) To optimize the conditions for the developed LAMP assays.

ii) To determine the sensitivity and specificity of the assays.

iii) To compare the LAMP assays with conventional PCR.

iv) To evaluate the LAMP assays on spiked blood samples.

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CHAPETER 2: LITERATURE REVIEW

2.1 Definition of nosocomial infection

Nosocomial infection (NI) is also known as hospital-acquired infection. This infection usually cause high morbidity and mortality rates in hospitals or healthcare facilities worldwide (Hughes et al., 2005). The etiologic agents of NI can spread easily in hospitals through the air, medical equipment or hands of healthcare workers and can cause infection because of the low immune system of hospitalized patients. Among all major complications that happened in the hospital, nosocomial infections take amount of 50%; the infection reasons are medication errors, patient falls, and other events (Becker et al., 1987). In the USA, there are roughly 1.7 million NI and 99,000 deaths each year (Pollack, 2010). In Europe, Gram-negative infections are estimated to two- thirds of the 25,000 deaths each year (Pollack, 2010). According to Hughes et al.

(2005), the rate of NI is 13.9% among 535 patients surveyed in the University of Malaya Medical Center. Bacterial agents, viruses, fungi, and parasites are recognized as sources of nosocomial infections, among them, bacterial agents are the most commonly recognized cause of hospital-acquired infections. Some of the nosocomial bacteria are antibiotic resistant and a majority of the antimicrobial resistance problems are typically associated with gram-positive nosocomial pathogens (Singh et al., 2006). There are lots of bacteria species in the group of nosocomial pathogens, and these include Enterococcus spp., Escherichia coli, Pseudomonas spp., Staphylococcus aureus.

Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii are

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three common pathogens that are often associated with NI.

2.2 Klebsiella pneumoniae

Klebsiella pneumoniae (K. pneumoniae) is an encapsulated, rod-shape, non-motile, Gram-negative bacterium of the family of Enterobacteriaceae, causes pneumonia, bacteremia and meningitis. It was first found in the sputum of lobar pneumonia in 1882 by Friedlande, so, it is also called Friedlande bacillus. K. pneumoniae is the most important member in the Klebsiella spp. About 95% of the Klebsiella infections are caused by K. pneumoniae. A survey on the data of an adult medical-surgical ICU ward of a University Hospital and two governmental hospitals in Malaysia from October 2003 to December 2006 showed that the most common causative pathogen was K.

pneumoniae (Katherason et al., 2009). K. pneumoniae is responsible for 4% - 8%

proportion for the respiratory NI (Diancourt et al., 2005). It can not only cause pneumonia, but also can cause urinary tract infection, biliary tract infection, septicemia and meningitis. This infection is more common among elderly, malnutrition, chronic alcoholism, and chronic bronchial-lung disease patients. A study in Malaysia and Japan estimated that the rate of this infection in elderly persons is 15%-40% (Umeh et al., 2002). K. pneumoniae often exists in human upper respiratory tract and intestinal tract, when the immunity of human body is reduced, the pathogen can go into the lungs through respiratory tract and cause big leaf lesions or lobular fusion. K. pneumoniae is the fourth or fifth most common cause of pneumonia and bacteremia, respectively (National Nosocomial Infections Surveillance (NNIS) System Report, 2003). This

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bacterium even have the cephalosporin resistance strains which caused an outbreak in New York City previously, this kind of strain is resistant to almost every common antibiotics and the control of this strain is very crucial (Bratu et al., 2005). The mortality due to pneumonia caused by K. pneumoniae is 50% (Umeh et al., 2002). For the detection of K. pneumoniae, there are a lot of published papers based on Polymer Chain Reaction (PCR). The genes that used for PCR primer target were rpoB (Chander et al., 2011), pehX (Kovtunovych et al., 2003), and gyrA (Brisse and Verhoef, 2001).

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 three 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).

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2.4 Acinetobacter baumannii

Acinetobacter baumannii (A. baumannii) is a Gram-negative bacterium, which 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 to 2009 in 36 countries, the resistance rates of A. baumannii are all above 50%

(Rosenthal et al,. 2012).

2.5 Conventional detection methods for Nosocomial pathogens

Nowadays, the treatment of NI is more difficult due the multidrug resistance property of these bacterial pathogens and the rapid spread of nosocomial pathogens will eventually increase the difficulty in treatment due to delayed detection and diagnosis.

Therefore, there is a need to develop a more rapid detection system to avoid further complications. Hence, different detection methods such as conventional culture method,

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PCR, real-time PCR and ELISA have been developed for the detection of nosocomial pathogens. A faster and accurate identification of the etiologic agents would be needed for prompt treatment of hospitalized patients.

Conventional culture method is time consuming and the results may be subjective, more than 1 day will be needed with this method. Now, PCR is the most common method applied in detection of bacterial pathogens. PCR is more sensitive, specific and faster compared to conventional blood culture methods. However, PCR involves multiple steps and requires special equipment such as the thermocycler and an electrophoretic system. Furthermore, this technique is also dependent on the skill of the person who performs PCR (Yamazaki, 2009). Another advanced diagnostic method, real-time PCR is more rapid and sensitive compared to PCR but requires an expensive thermal cycler with a fluorescence detector and the reagents are costly, and therefore, this method is limited diagnostic laboratories with sufficient resources (Mullah et al., 1998). For ELISA, the detection requires a high population of the target pathogen (Chapman et al., 2001).

2.6 Loop-mediated isothermal amplification (LAMP) method

An alternative method called the loop-mediated isothermal amplification (LAMP) was recently developed to circumvent the problems in identification and detection of specific bacteria. This method can detect the pathogens rapidly and effectively under isothermal condition (Notomi et al., 2000). The nucleotide will amplified in a fixed

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many types of structures in large amount (Hara-Kudo et al., 2006). At the beginning, this method can be done only with a heating block and does not need extra detection step as the product can be viewed visually (Allison, 2008). As the development of this method, LAMP machine has been produced, inside the machine there is a turbimeter, and can show the turbidity graph during the reaction. Also, the LAMP machine does not need a lot of space, and even small lab can have it easily.

LAMP assay include a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. The mechanism of LAMP method is shown in Figure 2.1. An inner primer (FIP) containing sequence of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer (F3) releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner (BIP) and outer primers (B3) that hybridize to the other end of the target, which produces a stem- loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 109 copies of target in less than an hour (Notomi et al., 2000).

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Figure 2.1: Principle of LAMP (adapted from Notomi et al., 2000)

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LAMP is extensively used in the detection of food-born pathogens (Song et al., 2006, Zhao et al., 2010), nosocomial pathogens (Hill et al., 2008, Lim et al., 2013), virus (Paride et al., 2005, Poon et al., 2006), and parasite (Kuboki et al., 2003).

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CHAPETER 3: METHODOLOGY

3.1 Materials

3.1.1 Bacterial strains

A total of 107 bacterial strains including 30 A. baumannii, 30 P. aeruginosa, 30 K.

pneumoniae, 3 Escherichia coli, 4 Shigella spp., 4 Vibrio spp., and 6 Salmonella spp.

were revived from the glycerol stocks. All the strains were obtained from the culture collection of the Laboratory of Biomedical Science and Molecular Microbiology, Institute of Graduate Studies, University of Malaya (Appendix 1). The purity of the strains was checked by streaking the culture on appropriate selective media. The P.

aeruginosa strains were checked by CHROMagar, A. baumannii and K. pneumoniae were checked by MacConkey agar.

3.1.2 Chemicals and reagents

All the information about Chemicals and the preparation of the growth media, buffers and reagent used in this study are listed in Appendix 2.

3.2 Methods

3.2.1 DNA template

DNA extraction was performed on the overnight culture by direct cell lysate method.

Briefly, a loopful of colonies was suspended in 100 µl double distill water. The

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suspension was boiled at 99℃ for 5 min, snapped cool on ice for 10 min and centrifuge at 13,400 rpm for 3 min. The supernatant was used as DNA template for LAMP analysis. And then, the DNA template was keep in fridge at -20℃.

3.2.2 Primer design

Prior to primer design, selected genes would be checked by BLAST (http:/www.ncbi.nih.gov) to minimize the similarity to the other species. The sequence of gltA (Assession no. NC_011595.1), tonB (NC_012731.1) and 16S rRNA-processing protein rimM (NC_009656.1) were retrieved from NCBI Genbank for the oligonucleotide primers design for A. baumannii, K. pneumoniae and P. aeruginosa, respectively. Four primers including one forward inner primer (FIP), one backward inner primer (BIP), one forward outer primer (F3) and one backward outer primer (B3) were designed by using Primer Explorer V4 (EIKEN CHEMICAL CO., LTD. Japan).

For P. aeruginosa, another two loop primers-LF, LP were generated. The specificity of the designed primers was determined by using insilico PCR (http://insilico.ehu.es/PCR/).

3.2.3 LAMP reaction

For A. baumannii: The reaction mixture in a total volume of 25 µl contained 12.5 µl

RM (EIKEN CHEMICAL CO.),40 pmol (each) of FIP and BIP, 5 pmol for each of F3 and B3, 1µl DNA polymerase and 2.5 µl DNA template. The reaction was incubated in

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Loopamp EXIA machine the real-time turbidimeter (EIKEN CHEMICAL CO., LTD), under 62 ℃ for 90 min and followed by inactivation at 80 ℃ for 2 min. Each run contained positive and negative control.

For K. pneumoniae: The reaction mixture in a total volume of 25 µl contained 12.5

µl RM (EIKEN CHEMICAL CO.),40 pmol (each) of FIP and BIP, 5 pmol for each of

F3 and B3, 1µl DNA polymerase and 2.5 µl DNA template. The reaction tube was then incubated in the Loopamp EXIA machine the real-time turbidimeter (EIKEN CHEMICAL CO., LTD), under 65 ℃ for 90 min and followed by inactivation at 80 ℃ for 2 min. Each run contained positive and negative controls.

For P. aeruginosa: The reaction mixture in a total volume of 25 µl contained 12.5 µl

RM (EIKEN CHEMICAL CO.),40 pmol (each) of FIP and BIP, 5 pmol for each of F3

and B3, 20 pmol (each) of LF and LB, 1µl DNA polymerase, 1 µl double distilled water and 2.5 µl DNA template. The reaction was incubate in Loopamp EXIA machine the real-time turbidimeter (EIKEN CHEMICAL CO., LTD), under 65 ℃ for 60 min and followed by inactivation at 80℃ for 2 min. Each run contained positive and negative controls.

3.2.4 Data analysis

The analysis of the LAMP product can be visualized by three methods:

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3.2.4.1 Real time monitoring by turbidity change

The visualization of the LAMP product was checked by turbidity. Positive results were indicated by the increase in turbidity above the threshold (0.1) within 70 min. No changes in turbidity indicated negative result with the Loopamp EXIA machine.

3.2.4.2 Formation of pellet method

The reaction tube was briefly spun for half a min. The presence of a pellet indicated a positive result, while the absence of a pellet indicated negative result.

3.2.4.3 Dye method

An aliquot of 1 µl of SYBR Green (10×dilution) was added into the LAMP product and the change of color from orange to green indicated a positive reaction, A negative result was indicated by no change in the orange color.

3.2.5 PCR

PCR was carried out in parallel with the LAMP assay to compare their specificity and sensitivity. PCR primers used for each of these three bacterial species are shown in Table 3.1. The target gene for PCR test of A. baumannii was blaOXA-51 gene since it was reported this gene is universally present in this bacterium (Turton et al., 2006). PCR reaction was carried out in a total volume of 25 µl, containing 1×PCR buffer, 1.2 mM MgCl2, 120 mM each dNTPs, 0.5 µM of each primer, 1 U of Taq DNA polymerase and 5 µl of DNA sample. The cycling conditions consisted of an initial denaturation at of

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94℃ for 5 min followed by 30 cycles of 25 s at 94℃, 40 s at 52℃, 50 s at 72℃ and the final extension at 6 min at 72℃.

For K. pneumoniae, the target gene was mdh (Thong et al., 2011). The PCR mix (25µl) consisted of 1×PCR buffer, 1.4 mM MgCl2, 140 mM each of the four nucleotides, 5 µl (20ng) DNA sample, 0.3 µM of each primer and 1U Taq DNA polymerase. The cycling conditions consisted of an initial denaturation at 95℃ for 5 min, 30 cycles of 95℃ for 1 min, 53℃ for 1min and 72℃ for 1 min, followed by an extension of 72℃ for 5 min.

For P. aeruginosa, the target gene was algD (Da et al., 1999) gene. The PCR mix (25µl) consisted of 1×PCR buffer, 2 mM MgCl2, 200 mM each of the four nucleotides, 5 µl (20ng) DNA sample, and 0.4 µM of each primer and 1U Taq DNA polymerase.

The cycling conditions consisted of an initial denaturation at 94℃ for 5 min, 30 cycles of 94℃ for 5 min, 60℃ for 1min and 72℃ for 1 min, followed by an extension of 72℃

for 7 min.

The PCR products were monitored by 1.5% agarose gel electrophoresis and then visualized under a UV transiluminator after staining with ethidium btomide (EtBr) for 30 min.

Table 3.1: The information of PCR primers

Gene Sequence reference

blaOXA-51 F: 5'TAATGCTTTGATCGGCCTTG3'

R: 5'TGGATTGCACTTCATCTTGG3'

Turton et al., 2006

Mdh F: 5'GCGTGGCGGTAGATCTAAGTCATA3' Thong et al., 2011

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algD F: 5'TTCCCTCGCAGAGAAAACATC3' R: 5'CCTGGTTGATCAGGTCGATCT3'

Da et al., 1999

3.2.6 Evaluation of sensitivity of LAMP assay in culture and blood samples

Culture sample: A colony of fresh bacterial culture was inoculated into 1 ml of LB

broth and incubated at 37℃ for 3 hours with agitation until the OD600 of the cell cultures reached approximately 1. Then a 10-fold dilution was done. An aliquot of 100 µl of each dilution was spread on both LB agar and selective media for the CFU count while another 100 µl was subjected to DNA extraction for LAMP and PCR assays.

Blood sample: A colony of fresh bacterial culture was inoculated into 1 ml of LB broth

and incubated at 37℃ for 3 hours with agitation until the OD600 of the broth reached approximately 1. An aliquot of 100 µl of the culture was spiked in 900 µl blood (healthy volunteer) and incubated at 37℃ for 2 h, followed by a 10-fold dilution. A 100 µl of each dilution was spread on both LB agar and selective media for the CFU count while another 100 µl was subjected to DNA extraction for LAMP and PCR assays.

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CHAPTER 4: RESULTS

4.1 Primer designed for LAMP

Suitable genes for primer design were found from the literature. More than five published papers about the detection of these three pathogens were found. After BLAST analysis, only a few genes were suitable. The LAMP primer design software showed 5 primers for each suitable gene fragment. These primers were then subjected to in silico test to determine specificity. The result of in silico is shown in Appendix 3-11. Due to each LAMP primer has two sets, the in silico was run twice to check the specificity. The nucleotide sequences of the primers were being filed for patent. The primers used for optimization were synthesized by a commercial company, BIONEER Company (Korea). For P. aeruginosa, an additional set of loop primer was synthesized to reduce the amplification time.

4.2 Acinetobacter baumannii 4.2.1 Optimized LAMP assay

The temperature and time of reaction were optimized for A. baumannii. Four different temperatures were tested for A. baumannii which included 60℃, 62℃, 63℃ and 65℃.

At 62℃, the assay took the shortest time to obtain amplification. Generally, the optimized temperature for A. baumannii was 62℃ (Fig 4.1) and the whole reaction time was fixed at 90 min. Fig 4.1 shows the temperature optimization for the positive control strain, AC081229. The curves were obtained directly from the screen of the Loopamp

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EXIA machine. The reaction amplification time for A. baumannii ranged from 60 to 80 min for different strains.

0 10 20 30 40 50 60 70 80 90 100

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

60℃

62℃

63℃

65℃

Fig 4.1: Comparison of LAMP results at different temperatures of A. baumannii

4.1.2 Sensitivity and specificity of the method

For analysis of the LAMP results, three methods were used for end point detection.

Firstly, when the reaction tube was briefly spun, a white pellet was formed at the bottom of the tube for the sample which showed positive amplification. For tube with negative amplification, no precipitate formed was observed. Secondly, a color change was observed when 1 µl of SYBR Green Ι was added to the reaction tube (Fig 4.2). Thirdly, when the amplification products were analyzed on agarose gel electrophoresis, a positive amplification was indicated by smears (multiple bands) (Fig 4.3). If there was no amplification, no band or smear was shown on the gel.

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The LAMP assay correctly identified 30 positive strains and no positive reaction was observed for all 20 non-A. baumannii strains with Loopamp EXIA machine. The non- A. baumannii strains were Klebsiella pneumoniae, E. coli, Vibrio, Shigella and Salmonella. In order to know the real specificity of the method, a comparison was made between LAMP and PCR. The results for these two methods are shown in Table 4.2.

Both the LAMP and PCR had the same specificity that all A. baumannii gave positive amplification while non-A. baumannii strains were not amplified.

Table 4.1: PCR and LAMP results of A. baumannii strains

Species/Subspecies Identification code

Specific

PCR results LAMP-result

Real Time Gel SYBR Green I

A. baumannii AC/060110 + + + +

A. baumannii AC/0612-17 + + + +

A. baumannii AC/0701-11 + + + +

A. baumannii AC/0702-5 + + + +

A. baumannii AC/0702-17 + + + +

A. baumannii AC/0703-14 + + + +

A. baumannii AC/0703-21 + + + +

A. baumannii AC/0711-7 + + + +

A. baumannii AC/0801-6 + + + +

A. baumannii AC/0801-11 + + + +

A. baumannii AC/0801-13 + + + +

A. baumannii AC/0802-1 + + + +

A. baumannii AC/0802-4 + + + +

A. baumannii AC/0802-14 + + + +

A. baumannii AC/0802-20 + + + +

A. baumannii AC/0803-15 + + + +

A. baumannii AC/0804-19 + + + +

A. baumannii AC/0812-29 + + + +

A. baumannii AC/0901-5 + + + +

A. baumannii AC/0901-14 + + + +

A. baumannii AC/0901-36 + + + +

A. baumannii AC/0901-37 + + + +

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A. baumannii AC/0902-6 + + + +

A. baumannii AC/0902-13 + + + +

A. baumannii AC/0902-14 + + + +

A. baumannii AC/0902-15 + + + +

A. baumannii AC/0903-15 + + + +

A. baumannii AC/0904-3 + + + +

A. baumannii AC/0905-31 + + + +

Klebsiella

pneumoniae PS81 - - - -

Klebsiella

pneumoniae PS92 - - - -

Klebsiella

pneumoniae PS88 - - - -

E. coli P49 - - - -

E. coli P41 - - - -

E. coli BS4 - - - -

Vibrio VPD21 - - - -

Vibrio VPD22 - - - -

Vibrio VPD26 - - - -

Vibrio VPD27 - - - -

Shigella flexneri TH32/98 - - - -

Shigella sonnei TC3/99 - - - -

Shigella flexmeri TH23/97 - - - -

Shigella flexneri Y

variant TH5/02 - - - -

Salmonella S.Meto303/94 - - - -

Salmonella S.Oke-nara - - - -

Salmonella S.LOM - - - -

Salmonella S.Bevis - - - -

Salmonella S.Hvrt - - - -

Salmonella SAB79 - - - -

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Fig 4.2: Detection of the LAMP products using SYBR Green Ι.

N indicates negative sample, P indicates positive sample.

Negative result is indicated by no color change while positive result is indicated by a color change to green.

Fig 4.3: Visualization of LAMP products by agarose gel electrophoresis.

Lane L, 100-bp DNA ladder; Lane N, negative control; Lane 1, AC071107; Lane 2, AC081229; Lane 3, AC090203; Lane 4, AC090215; Lane 5, BS4 (E. coli); Lane 6, p41(

E. coli) ; Lane 7, p49( E. coli) ; Lane 8 PS81 (K. pneumoniae); Lane 9, PS88. (K.

pneumoniae).

A positive LAMP result is indicated by smearing (lanes 1-4) while negative results have no amplification (lanes 5-9).

P N P

N

9 9

L 8

7 6 5 4 2 3

N 1

L

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In order to know the sensitivity or the detection limit of the LAMP assay, a parallel test with PCR was carried out. DNA from an aliquot of each dilution was tested with the LAMP and PCR. The detection limit of LAMP assay and PCR was 5.5×104 CFU/ml (equal to 1375 CFU per reaction) and 5×105 CFU/ml, respectively. This shows that the LAMP method for detection of A.baumannii was 10-fold more sensitive than PCR (Fig 4.4).

(a) 0 20 40 60 80 100 120

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

1 2 3 4 5

(b)

Fig 4.4: Sensitivity of the LAMP assay (a) and PCR (b) for A. baumannii AC090215.

1=5.5×107cfu/ml; 2=5.5×106; 3=5.5×105cfu/ml; 4=5.5×104cfu/ml; 5=5.5×104cfu/ml.

Fig (a): Amplified product with LAMP is seen for lanes 1 - 4.

Fig (b): Amplified product with PCR is seen for lanes 1 - 3.

1 2 4 5

353bp

Ladder 3

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4.1.3Evaluation of LAMP on Spiked blood sample

The performance of PCR and LAMP assay on spiked blood samples was comparable.

Blood specimen (from the author) was taken by a doctor at the student and staff clinic of University of Malaya. The detection limit of A. baumannii for both PCR and LAMP assays was 5.5×105 CFU/ml which equals to 1100 CFU per reaction.

(a) 0 20 40 60 80 100 120

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

1 2 3

(b)

Fig 4.5: The detection limit of LAMP (a) and PCR (b) using A. baumannii spiked blood sample.

3 353bp

2 1

Ladder

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Amplified product is seen for lanes 1-2.

4.2 P. aeruginosa

4.2.1 Optimized LAMP assay

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.

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0 10 20 30 40 50 60 70 80 90 0

0.1 0.2 0.3 0.4 0.5

0.6 60

62 63 64

Fig 4.6: Comparison of LAMP results for P. aeruginosa PS19 at different temperatures

0 10 20 30 40 50 60 70 80 90 100

0 0.1 0.2 0.3 0.4 0.5 0.6

with loop primer without loop primer

Fig 4.7: Comparison of LAMP results with or without loop primer on PS19

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4.2.2 Sensitivity and specificity of the method

In order to test the specificity of the assay, 50 bacterial cultures were used. Positive reaction was observed with 30 P. aeruginosa strains while no amplification was observed for the 20 non-P. aeruginosa strains which included Klebsiella pneumoniae, E. coli, Vibrio, Shigella and Salmonella.

PCR was also used on all of these strains. Table 4.2 shows the results of these two methods. Both the LAMP and PCR had the same specificity, in which all P. aeruginosa gave positive amplification while non- P. aeruginosa strains were not amplified.

Table 4.2: PCR and LAMP result of P. aeruginosa strains

Species/Subspecies Identification code

Specific

PCR results LAMP-result

Real Time Gel SYBR Green I

P. aeruginosa PS2 + + + +

P. aeruginosa PS16 + + + +

P. aeruginosa PS19 + + + +

P. aeruginosa PS20 + + + +

P. aeruginosa PS23 + + + +

P. aeruginosa PS67 + + + +

P. aeruginosa PS98 + + + +

P. aeruginosa PS100 + + + +

P. aeruginosa PS102 + + + +

P. aeruginosa PS103 + + + +

P. aeruginosa PS105 + + + +

P. aeruginosa PS108 + + + +

P. aeruginosa PS110 + + + +

P. aeruginosa PS239 + + + +

P. aeruginosa PS339 + + + +

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P. aeruginosa PS341 + + + +

P. aeruginosa PS362 + + + +

P. aeruginosa R01 + + + +

P. aeruginosa R02 + + + +

P. aeruginosa R04 + + + +

P. aeruginosa 1182 + + + +

P. aeruginosa 1186 + + + +

P. aeruginosa 1260 + + + +

P. aeruginosa 1288 + + + +

P. aeruginosa B14141 + + + +

P. aeruginosa B14143 + + + +

P. aeruginosa B14128 + + + +

P. aeruginosa B14262 + + + +

P. aeruginosa B14349 + + + +

P. aeruginosa BF2087 + + + +

Klebsiella

pneumoniae PS81 - - - -

Klebsiella

pneumoniae PS92 - - - -

Klebsiella

pneumoniae PS88 - - - -

E. coli P49 - - - -

E. coli P41 - - - -

E. coli BS4 - - - -

Vibrio VPD21 - - - -

Vibrio VPD22 - - - -

Vibrio VPD26 - - - -

Vibrio VPD27 - - - -

Shigella flexneri TH32/98 - - - -

Shigella sonnei TC3/99 - - - -

Shigella flexneri TH23/97 - - - -

shigella flexneri Y

variant TH5/02 - - - -

Salmonella S.Meto303/94 - - - -

Salmonella S.Oke-nara - - - -

Salmonella S.LOM - - - -

Salmonella S.Bevis - - - -

Salmonella S.Hvrt - - - -

Salmonella SAB79 - - - -

The detection limit of this assay on bacterial culture was 3.6×104 CFU/ml which

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CFU/ml. So, this LAMP assay was 1000-fold more sensitive than conventional PCR (Fig 4.8).The sensitivity test was based on the 10-fold serial dilution, and the initial inoculum of P. aeruginosa was 3.6×106 CFU/ml.

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(a) 0 10 20 30 40 50 60

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45

1 2 3 4 5

(b)

Fig 4.8: Sensitivity of the LAMP assay (a) and PCR (b) for P. aeruginosa.PS19.

1 = 3.6×107 CFU/ml; 2 = 3.6×106 CFU/ml; 3 = 3.6×105 CFU/ml; 4 = 3.6×104 CFU/ml; 5

= 3.6×103 CFU/ml; N = negative control (distilled water).

Fig (a): Amplified product is seen for lanes 1-4.

Fig (b): Amplified product is seen for lanes 1.

4.2.3Evaluation of LAMP on spiked blood

In order to check the sensitivity of the LAMP, the test was carried out with DNA prepared serially diluted spiked blood samples. The detection limit of LAMP assay for P. aeruginosa was 7.7×104 CFU/ml which means 154 CFU per reaction (Fig 4.9). The 520bp

5 3 4

Ladder

1 2

N

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detection limit for PCR was 7.7×107 CFU/ml. Therefore, the sensitivity of LAMP assay for P. aeruginosa was much higher than conventional PCR.

(a) 0 10 20 30 40 50 60 70

-0.01 0.04 0.09 0.14 0.19 0.24 0.29 0.34 0.39 0.44 0.49

1 2 3 4 5 6

(b)

Fig 4.9: The detection limit of LAMP (a) and PCR (b) using P. aeruginosa spiked blood sample.

Fig (a): 1 = negative control (distilled water); 2 = 7.7×107 CFU/ml; 3 = 7.7×106 CFU/ml;

4 = 7.7×105 CFU/ml; 5 = 7.7×104 CFU/ml; 6 = 7.7×103 CFU/ml.

Amplified product is seen for lanes 2-5.

Fig (b): N = negative control (distilled water); 1 = 7.7×107 CFU/ml; 2 = 7.7×106 CFU/ml; 3 = 7.7×105 CFU/ml; 4 = 7.7×104 CFU/ml; 5 = 7.7×103 CFU/ml.

Amplified product is seen for lanes 1.

520bp

1 2 3 4 5

N Ladder

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4.3 K. pneumoniae

4.3.1 Optimized LAMP assay

Five temperatures were tested to determine the optimum temperature. These included 60℃, 62℃, 63℃, 64℃ and 65℃. At 65℃, the reaction had the shortest amplification time. Fig 4.10 shows the turbidity curve of the reaction which amplified at 65℃. No loop primer was designed for K. pneumoniae LAMP assay, because the amplification time was less than 60 min.

0 10 20 30 40 50 60 70 80 90 100

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Fig 4.10: Optimized temperature (65℃) for K. pneumoniae LAMP detection

4.3.2 Sensitivity and specificity of the method

In order to test the specificity of the assay, 50 bacterial cultures were used. Positive reaction was observed with 30 K. pneumoniae strains while no amplification was observed for the 20 non- K. pneumoniae strains which included P. aeruginosa, E. coli,

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PCR was also used on all of these strains. Table 4.3 shows the result of these two methods. All the LAMP results were confirmed by three different methods, there were turbidity method, fluorescence (SYBR Green 1) and precipitation. The results showed that both LAMP and PCR had highly specificity in the detection of P. aeruginosa.

Table 4.3: Comparison of PCR and LAMP result of K. pneumoniae strains

Species/Subspecies Identification code

Specific

PCR results LAMP-result

Real Time Gel SYBR Green I

K. pneumoniae PS19 + + + +

K. pneumoniae PS23 + + + +

K. pneumoniae PS31 + + + +

K. pneumoniae PS35 + + + +

K. pneumoniae PS36 + + + +

K. pneumoniae PS49 + + + +

K. pneumoniae PS50 + + + +

K. pneumoniae PS51 + + + +

K. pneumoniae PS80 + + + +

K. pneumoniae PS82 + + + +

K. pneumoniae PS83 + + + +

K. pneumoniae PS86 + + + +

K. pneumoniae PS88 + + + +

K. pneumoniae PS90 + + + +

K. pneumoniae PS92 + + + +

K. pneumoniae PS96 + + + +

K. pneumoniae PS138 + + + +

K. pneumoniae PS156 + + + +

K. pneumoniae K09-24 + + + +

K. pneumoniae K09-25 + + + +

K. pneumoniae K10-03 + + + +

K. pneumoniae K10-04 + + + +

K. pneumoniae K10-05 + + + +

K. pneumoniae K11-01 + + + +

K. pneumoniae K11-02 + + + +

K. pneumoniae K11-03 + + + +

K. pneumoniae K11-04 + + + +

K. pneumoniae K11-05 - - - -

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