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Establishment of rapid detection of Haemophilus influenzae type b by isothermal amplification of bexA gene

Haemophilus influenzae type b (Hib) is a common cause of childhood meningitis that leads to a significant morbidity and mortality in both developing countries and underdeveloped countries, where the successful Hib conjugate vaccine is not accessible. A rapid and costeffective diagnostic method wa...

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Main Author: Siong, Leow Chee
Format: Monograph
Language:English
Published: Pusat Pengajian Kesihatan 2015
Subjects:
RA0421 Public health. Hygiene. Preventive Medicine
RC109-216 Infectious and parasitic diseases
Online Access:http://eprints.usm.my/60006/1/LEOW%20CHEE%20SIONG%20-%20e.pdf
http://eprints.usm.my/60006/
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spelling my.usm.eprints.60006 http://eprints.usm.my/60006/ Establishment of rapid detection of Haemophilus influenzae type b by isothermal amplification of bexA gene Siong, Leow Chee RA0421 Public health. Hygiene. Preventive Medicine RC109-216 Infectious and parasitic diseases Haemophilus influenzae type b (Hib) is a common cause of childhood meningitis that leads to a significant morbidity and mortality in both developing countries and underdeveloped countries, where the successful Hib conjugate vaccine is not accessible. A rapid and costeffective diagnostic method was highly demanded in resource-limited laboratory. Hence, the aim of this study is to establish a rapid detection method for H. influenzae type b by loopmediated isothermal amplification (LAMP) of bexA gene. A LAMP primer set was specifically designed to target bex\ gene. An in-house LAMP reaction was established and optimized using extracted genomic DNA of Haemophilus influenzae ATCC 10211. Subsequently, both LAMP reagents and reaction conditions were optimized and the optimized LAMP assay was subjected to analytical specificity and sensitivity evaluation. PCR reaction was conducted by using outer primers to provide comparison in term of analytical sensitivity. The analytical specificity was evaluated with thirty non-haemophilus strains which showed 100% specificity for bexX gene identification. The limit of detection (LOD) for LAMP assay was determined at 2 pg of DNA per reaction and as low as 2 colonies-forming units (CFU) per reaction. This indicates that the detection limit of LAMP by CFU per reaction was 100 fold lower than the detection limit of PCR. Based on the result obtained, LAMP has the potential to become an excellent diagnostic tool capable to produce a rapid, highly sensitive and specific result in a single temperature condition. Pusat Pengajian Kesihatan 2015 Monograph NonPeerReviewed application/pdf en http://eprints.usm.my/60006/1/LEOW%20CHEE%20SIONG%20-%20e.pdf Siong, Leow Chee (2015) Establishment of rapid detection of Haemophilus influenzae type b by isothermal amplification of bexA gene. Project Report. Pusat Pengajian Kesihatan. (Submitted)
institution Universiti Sains Malaysia
building Hamzah Sendut Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Sains Malaysia
content_source USM Institutional Repository
url_provider http://eprints.usm.my/
language English
topic RA0421 Public health. Hygiene. Preventive Medicine
RC109-216 Infectious and parasitic diseases
spellingShingle RA0421 Public health. Hygiene. Preventive Medicine
RC109-216 Infectious and parasitic diseases
Siong, Leow Chee
Establishment of rapid detection of Haemophilus influenzae type b by isothermal amplification of bexA gene
description Haemophilus influenzae type b (Hib) is a common cause of childhood meningitis that leads to a significant morbidity and mortality in both developing countries and underdeveloped countries, where the successful Hib conjugate vaccine is not accessible. A rapid and costeffective diagnostic method was highly demanded in resource-limited laboratory. Hence, the aim of this study is to establish a rapid detection method for H. influenzae type b by loopmediated isothermal amplification (LAMP) of bexA gene. A LAMP primer set was specifically designed to target bex\ gene. An in-house LAMP reaction was established and optimized using extracted genomic DNA of Haemophilus influenzae ATCC 10211. Subsequently, both LAMP reagents and reaction conditions were optimized and the optimized LAMP assay was subjected to analytical specificity and sensitivity evaluation. PCR reaction was conducted by using outer primers to provide comparison in term of analytical sensitivity. The analytical specificity was evaluated with thirty non-haemophilus strains which showed 100% specificity for bexX gene identification. The limit of detection (LOD) for LAMP assay was determined at 2 pg of DNA per reaction and as low as 2 colonies-forming units (CFU) per reaction. This indicates that the detection limit of LAMP by CFU per reaction was 100 fold lower than the detection limit of PCR. Based on the result obtained, LAMP has the potential to become an excellent diagnostic tool capable to produce a rapid, highly sensitive and specific result in a single temperature condition.
format Monograph
author Siong, Leow Chee
author_facet Siong, Leow Chee
author_sort Siong, Leow Chee
title Establishment of rapid detection of Haemophilus influenzae type b by isothermal amplification of bexA gene
title_short Establishment of rapid detection of Haemophilus influenzae type b by isothermal amplification of bexA gene
title_full Establishment of rapid detection of Haemophilus influenzae type b by isothermal amplification of bexA gene
title_fullStr Establishment of rapid detection of Haemophilus influenzae type b by isothermal amplification of bexA gene
title_full_unstemmed Establishment of rapid detection of Haemophilus influenzae type b by isothermal amplification of bexA gene
title_sort establishment of rapid detection of haemophilus influenzae type b by isothermal amplification of bexa gene
publisher Pusat Pengajian Kesihatan
publishDate 2015
url http://eprints.usm.my/60006/1/LEOW%20CHEE%20SIONG%20-%20e.pdf
http://eprints.usm.my/60006/
_version_ 1797907841546190848
score 13.211869

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