| Author: | Lam, Siu-bing Kendra |
| Title: | An inhibitor-controlled real-time PCR assay for direct detection of Vibrio parahaemolyticus in stool |
| Degree: | M.Sc. |
| Year: | 2009 |
| Subject: | Hong Kong Polytechnic University -- Dissertations. Vibrio infections. Vibrio parahaemolyticus. Polymerase chain reaction. |
| Department: | Department of Health Technology and Informatics |
| Pages: | xi, 58 leaves : ill. (some col.) ; 30 cm. |
| Language: | English |
| Abstract: | Vibrio parahaemolyticus (V. parahaemolyticus) is the commonest cause of Vibrio infections worldwide and it is the top causative agent among all the reported food poisoning outbreaks. The usefulness of real-tune PCR as a means of detecting toxR gene in stool samples from patients with diarrhea was studied. Simple boiling extraction and commercial kit (QIAamp DNA Stool Mini Kit, Qiagen) extraction methods were compared, with conventional culture method as the gold standard. An internal amplification control was included so as to identify any false negative PCR results. The limit of detection (LoD) in this assay was determined as 104 CFUml-1 of V. parahaemolyticus in stool with the mean crossing point value at 29.0. Of the 53 V. parahaemolyticus culture-positive and 39 V, parahaemolyticus culture-negative specimens, 33 specimens with simple boiling extraction were inhibited in the real-time PCR that accounted for 36%. The sensitivity and of boiling extracts were 69%. For the real-time PCR results of kit extracts, no inhibition was detected that demonstrated the effective removal of the inhibitory substance by the QIAamp DNA Stool Mini Kit (Qiagen). The sensitivity and specificity of the real-tune PCR with kit extraction were 100% and 72%, respectively when compared with the culture method. The relatively low specificity was due to the results of V. parahaemolyticus culture-negative but real-time PCR-positive samples. These samples were further investigated with the sequencing technique and the results showed that they were V. parahaemolyticus with 98-100% resemblance to the species. These negative V. parahaemolyticus culture results might be due to the viable but non-cultural state of the organisms or the lower sensitivity of culture method. The relative high crossing point value of these specimens (mean Cp=32.8, n=l1) meant they contained the toxR sequence DNA was below the LoD of this assay (l04 CFU ml-1 of V. parahaemolyticus in stool, Cp=29.0). Since these samples had a high degree of variability and might not be reproducible, these results should be interpreted with caution. If these unambiguous results were excluded from calculation, the specificity and positive predictive value of the real-time PCR with kit extraction compared to culture method would become 100%. |
| Rights: | All rights reserved |
| Access: | restricted access |
Files in This Item:
| File | Description | Size | Format | |
|---|---|---|---|---|
| b23056101.pdf | For All Users (off-campus access for PolyU Staff & Students only) | 4.13 MB | Adobe PDF | View/Open |
Copyright Undertaking
As a bona fide Library user, I declare that:
- I will abide by the rules and legal ordinances governing copyright regarding the use of the Database.
- 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.
- 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.
Please use this identifier to cite or link to this item:
https://theses.lib.polyu.edu.hk/handle/200/4769