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Seroprevalence And Molecular Detection Of Brucellosis In Hospitalized Patients With Clinical Manifestations Of Brucellosis

By: Riffat Yousaf (2008-VA-342) | Dr. Wasim Shehzad.
Contributor(s): Prof. Dr.Tahir Yaqub | Dr. Haroon Akbar.
Material type: materialTypeLabelBookPublisher: 2015Description: 64p.Subject(s): Institute of Biochemistry and Biotechnology | Molecular Biology and BiotechnologyDDC classification: 2286-T Dissertation note: Brucella species are host specific facultative intracellular pathogens which cause brucellosis in both animals and humans. Brucellosis is one of the most common zoonotic diseases worldwide. In Pakistan, the incidence of brucellosis is increasing day by day due to lack of awareness of this deadly malady. It is transmitted from infected animals to humans who are in close contact with infected vaginal secretions, feces, blood, aborted fetus, or by consumption of unpasteurized milk and dairy products. Infection due to B. melitensis and B. abortus are mostly prevalent for brucellosis in human. Total 218 blood samples were collected in gel vacutainer tubes from hospitalized patients who were clinically manifested with brucellosis. Out of 218, 12 RBPT positive blood samples were collected in EDTA containing vacutainer tubes separately. Serum was isolated from all blood samples (without EDTA). These serum samples were first screened by Rose Bengal Plate Test (RBPT). DNA was extracted from all positive RBPT blood and serum samples and randomly selected negative RBPT serum samples. All extracted DNA (≤10ng/µL) were subjected to Brucella genus and two species specific (B. abortus and B. melitensis) Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) assay. Furthermore, few selected extracted DNA (≥20ng/µL) from blood and serum samples were examined by genus and Multiplex specie specific PCR. The PCR products were electrophoresed on 2.5% agarose gel. Then selected products were sequenced by ABI 3130 XL sequencer. The data were analyzed by SPSS software using Chi square test. The present study helped to diagnose accurately and precisely brucellosis in clinical manifested patients, which is further helpful for devising the strategies to control this disease.
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Brucella species are host specific facultative intracellular pathogens which cause brucellosis in both animals and humans. Brucellosis is one of the most common zoonotic diseases worldwide. In Pakistan, the incidence of brucellosis is increasing day by day due to lack of awareness of this deadly malady. It is transmitted from infected animals to humans who are in close contact with infected vaginal secretions, feces, blood, aborted fetus, or by consumption of unpasteurized milk and dairy products. Infection due to B. melitensis and B. abortus are mostly prevalent for brucellosis in human.
Total 218 blood samples were collected in gel vacutainer tubes from hospitalized patients who were clinically manifested with brucellosis. Out of 218, 12 RBPT positive blood samples were collected in EDTA containing vacutainer tubes separately. Serum was isolated from all blood samples (without EDTA). These serum samples were first screened by Rose Bengal Plate Test (RBPT). DNA was extracted from all positive RBPT blood and serum samples and randomly selected negative RBPT serum samples. All extracted DNA (≤10ng/µL) were subjected to Brucella genus and two species specific (B. abortus and B. melitensis) Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) assay. Furthermore, few selected extracted DNA (≥20ng/µL) from blood and serum samples were examined by genus and Multiplex specie specific PCR. The PCR products were electrophoresed on 2.5% agarose gel. Then selected products were sequenced by ABI 3130 XL sequencer. The data were analyzed by SPSS software using Chi square test.
The present study helped to diagnose accurately and precisely brucellosis in clinical manifested patients, which is further helpful for devising the strategies to control this disease.

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