Development Of A Rapid Molecular-Based Diagnostic Kit For Diagnosis Of Leptospirosis

Leptospirosis is a worldwide zoonosis, affecting farm animals, wildlife as well as humans particularly in tropical areas. It is a re-emerging infectious disease caused by pathogenic leptospires. Diagnosis of leptospirosis usually depends upon demonstration of serum antibodies by serological tests. T...

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Main Author:	Somkuna, Narumon
Format:	Thesis
Language:	English English
Published:	2004
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Online Access:	http://psasir.upm.edu.my/id/eprint/6427/1/ABSTRACT__FPV_2004_9.pdf
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spelling	my-upm-ir.64272013-05-27T07:29:28Z Development Of A Rapid Molecular-Based Diagnostic Kit For Diagnosis Of Leptospirosis 2004 Somkuna, Narumon Leptospirosis is a worldwide zoonosis, affecting farm animals, wildlife as well as humans particularly in tropical areas. It is a re-emerging infectious disease caused by pathogenic leptospires. Diagnosis of leptospirosis usually depends upon demonstration of serum antibodies by serological tests. These tests are relatively not specific, sensitive and often time-consuming. Early, quick and precise diagnosis is essential so that early and specific treatment of patients can be provided and also allows for suitable control of infection to be implemented. The primary objective of this study to use a polymerase chain reaction (PCR) assay as a major technique to detect and differentiate pathogenic leptospires. In addition, another aim of this study was to develop a diagnostic kit based on PCR assay coupled with DNA hybridization assay. The assay used a nonradioactive leptospiral DNA probe hybridized to target leptospiral DNA in a microtitre plate for the detection of leptospiral DNA in clinical specimens. Twenty-one reference leptospiral serovars representing four species of Leptospira (L. interrogans, L. weilii, L. inadai and L. borgpetersenii) and clinical samples were examined. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Low-Stringency-PCR (LS-PCR) were applied to detect and differentiate the leptospires. The results from PCR-RFLP showed that restriction enzymes: DdeI, NdeII, and MspI could digest the amplified leptospiral DNA with genus specific primers G1/G2 and allowed differentiation of the leptospiral serovars and serogroups into their species level. It is seen that LS-PCR could produce serovar specific DNA fingerprintings. Nonradioactive digoxigenin (DIG)-labelled leptospiral DNA probes and dot blot hybridization (DBH) assay were successfully developed for the detection of leptospiral DNA in cultures and clinical samples. Results showed that these probes had a high sensitivity and specificity. The lowest limit of the DNA concentration in PCR-DBH assay was 10-6 μg. A new and rapid diagnostic kit was thus designed. This PCR-MHA detection method has advantages over the conventional PCR assay and DBH assay when handling a large number of samples. The procedure has fewer steps and it is fast, safe and easy to handle. Results showed it has high specificity, sensitivity and reproducibility. Furthermore, the assay could be used to detect leptospiral DNA using reagents and standard laboratory ELISA equipment. Moreover, the entire method could be completed within a day. The technique could be directly applied to clinical samples such as urine and serum for rapid diagnosis of leptospirosis. The technique used in this study will be useful for detection and identification of leptospirosis in patients. Moreover, it could also be used on farm animals for screening and detection of uninfected animals. 2004 Thesis http://psasir.upm.edu.my/id/eprint/6427/ http://psasir.upm.edu.my/id/eprint/6427/1/ABSTRACT__FPV_2004_9.pdf application/pdf en public phd doctoral Universiti Putra Malaysia Faculty Veterinary Medicine English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
topic
spellingShingle	Somkuna, Narumon Development Of A Rapid Molecular-Based Diagnostic Kit For Diagnosis Of Leptospirosis
description	Leptospirosis is a worldwide zoonosis, affecting farm animals, wildlife as well as humans particularly in tropical areas. It is a re-emerging infectious disease caused by pathogenic leptospires. Diagnosis of leptospirosis usually depends upon demonstration of serum antibodies by serological tests. These tests are relatively not specific, sensitive and often time-consuming. Early, quick and precise diagnosis is essential so that early and specific treatment of patients can be provided and also allows for suitable control of infection to be implemented. The primary objective of this study to use a polymerase chain reaction (PCR) assay as a major technique to detect and differentiate pathogenic leptospires. In addition, another aim of this study was to develop a diagnostic kit based on PCR assay coupled with DNA hybridization assay. The assay used a nonradioactive leptospiral DNA probe hybridized to target leptospiral DNA in a microtitre plate for the detection of leptospiral DNA in clinical specimens. Twenty-one reference leptospiral serovars representing four species of Leptospira (L. interrogans, L. weilii, L. inadai and L. borgpetersenii) and clinical samples were examined. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Low-Stringency-PCR (LS-PCR) were applied to detect and differentiate the leptospires. The results from PCR-RFLP showed that restriction enzymes: DdeI, NdeII, and MspI could digest the amplified leptospiral DNA with genus specific primers G1/G2 and allowed differentiation of the leptospiral serovars and serogroups into their species level. It is seen that LS-PCR could produce serovar specific DNA fingerprintings. Nonradioactive digoxigenin (DIG)-labelled leptospiral DNA probes and dot blot hybridization (DBH) assay were successfully developed for the detection of leptospiral DNA in cultures and clinical samples. Results showed that these probes had a high sensitivity and specificity. The lowest limit of the DNA concentration in PCR-DBH assay was 10-6 μg. A new and rapid diagnostic kit was thus designed. This PCR-MHA detection method has advantages over the conventional PCR assay and DBH assay when handling a large number of samples. The procedure has fewer steps and it is fast, safe and easy to handle. Results showed it has high specificity, sensitivity and reproducibility. Furthermore, the assay could be used to detect leptospiral DNA using reagents and standard laboratory ELISA equipment. Moreover, the entire method could be completed within a day. The technique could be directly applied to clinical samples such as urine and serum for rapid diagnosis of leptospirosis. The technique used in this study will be useful for detection and identification of leptospirosis in patients. Moreover, it could also be used on farm animals for screening and detection of uninfected animals.
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Somkuna, Narumon
author_facet	Somkuna, Narumon
author_sort	Somkuna, Narumon
title	Development Of A Rapid Molecular-Based Diagnostic Kit For Diagnosis Of Leptospirosis
title_short	Development Of A Rapid Molecular-Based Diagnostic Kit For Diagnosis Of Leptospirosis
title_full	Development Of A Rapid Molecular-Based Diagnostic Kit For Diagnosis Of Leptospirosis
title_fullStr	Development Of A Rapid Molecular-Based Diagnostic Kit For Diagnosis Of Leptospirosis
title_full_unstemmed	Development Of A Rapid Molecular-Based Diagnostic Kit For Diagnosis Of Leptospirosis
title_sort	development of a rapid molecular-based diagnostic kit for diagnosis of leptospirosis
granting_institution	Universiti Putra Malaysia
granting_department	Faculty Veterinary Medicine
publishDate	2004
url	http://psasir.upm.edu.my/id/eprint/6427/1/ABSTRACT__FPV_2004_9.pdf
_version_	1747810609803558912

Development Of A Rapid Molecular-Based Diagnostic Kit For Diagnosis Of Leptospirosis

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