An inhibitor-controlled real-time PCR assay for direct detection of Vibrio parahaemolyticus in stool

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An inhibitor-controlled real-time PCR assay for direct detection of Vibrio parahaemolyticus in stool


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: Dept. 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%.

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