Detection and identification of four common human-infecting Plasmodium species by fluorescent hydrolysis probe-based real-time PCR technology

Pao Yue-kong Library Electronic Theses Database

Detection and identification of four common human-infecting Plasmodium species by fluorescent hydrolysis probe-based real-time PCR technology

 

Author: Chan, Ka Yan
Title: Detection and identification of four common human-infecting Plasmodium species by fluorescent hydrolysis probe-based real-time PCR technology
Degree: M.Sc.
Year: 2013
Subject: Plasmodium.
Polymerase chain reaction -- Diagnostic use.
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Health Technology and Informatics
Pages: xv, 125 leaves : col. ill., 1 col. map ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2637226
URI: http://theses.lib.polyu.edu.hk/handle/200/7063
Abstract: Introduction: Malaria is a mosquito-borne infectious disease caused by a parasite called Plasmodium. Five Plasmodium species infecting humans to cause malaria are P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi. Malaria affects approximately 3 billion people in 106 countries and territories from South America to the Indian peninsula with an estimated 250 million clinical cases and about 800,000 deaths annually. Rapid and accurate species identification in the diagnosis of malaria is important since the unique 48-72 hours asexual replication cycle among human and malaria parasites can rapidly result in high levels of parasitaemia with a fatal outcome in humans, especially in the most virulent P. falciparum infection. Microscopic examination and polymerase chain reaction (PCR)-based methods are commonly used in diagnosis of malaria. Microscopic examination of Giemsa stained blood films is the most widely practiced and useful method for definitive malaria diagnosis but microscopists need to be trained and supervised to ensure consistent reliability. Furthermore, inability to detect low levels of parasitaemia and mixed infections and risks of misdiagnosis are the limitations of microscopic examination in malaria diagnosis. Nowadays, PCR techniques are becoming a more frequently used tool in the diagnosis of malaria due to its extremely high sensitivity and specificity. The amplification-based, nested PCR method is an ideal assay capable of detecting parasitaemia with extremely low levels. However, this assay is labor intensive and long turn-around times, requiring up to 12 hours for processing as well as failures to offer quantitative result. Therefore, the demand for a more sensitive and time-efficient assay promotes the development of quantitative real-time PCR assays.
Aims: In the study, a reliable and rapid assay using sequence-specific primers and fluorescent hydrolysis probes was designed and optimized for detection and identification of Plasmodium species by LightCycler® version 2.0. Method: Archived samples from 2006-2012 extracted by magnetic based method were participated in this study. The selected samples were confirmed with Plasmodium infections by microscopy and nested PCR. The condition of multiple real-time PCR assay for identification of Plasmodium species was optimised by determination of variation in CT values for individual Plasmodium species in several trials and using gel electrophoresis to confirm the target products. Moreover, total of 105 samples were assessed by the multiplex real-time PCR assay and microscopy as gold standard. Key clinical parameters such as dynamic range, clinical and analytical sensitivity and specificity on samples and cost and turn-around-time analysis were evaluated. Results: The dynamic ranges of the assay for species identification of P. falciparum, P. vivax, P. malariae and P. ovale were 10¹¹-10³, 10¹¹-10³, 10¹¹-10⁵ and 10¹¹-10⁶ DNA copies/μL of sample DNA, respectively. The PCR efficiencies of species identification for the four Plasmodium species, P. falciparum, P. vivax, P. malariae and P, ovale were 1.8, 1.6, 1.9 and 1.6, respectively. Moreover, compared to microscopy as the reference standard, the real-time assay had a sensitivity of 100% and specificity of 100% for the identification of Plasmodium spp. Conclusion: Multiplex real-time PCR assay with fluorescent hydrolysis probe-based technology by LightCycler® version 2.0 showed high sensitivity and specificity for identification of four common Plasmodium species. This assay could probably provide a rapid and reliable platform for the detection and identification of four common human-infecting Plasmodium species infections and could substitute for the conventional nested PCR assay for applying in conjunction with microscopy to further confirmation of parasite infection.

Files in this item

Files Size Format
b26372265.pdf 4.187Mb PDF
Copyright Undertaking
As a bona fide Library user, I declare that:
  1. I will abide by the rules and legal ordinances governing copyright regarding the use of the Database.
  2. I will use the Database for the purpose of my research or private study only and not for circulation or further reproduction or any other purpose.
  3. I agree to indemnify and hold the University harmless from and against any loss, damage, cost, liability or expenses arising from copyright infringement or unauthorized usage.
By downloading any item(s) listed above, you acknowledge that you have read and understood the copyright undertaking as stated above, and agree to be bound by all of its terms.

     

Quick Search

Browse

More Information