Detection of viable Vibrio cholerae by reverse-transcriptase polymerase chain reaction (RT-PCR).
The use of conventional PCR can amplify target DNA from both viable and nonviable cells of Vibrio cholera. Detection of only viable microbial pathogens in biological samples, especially clinical and food samples, is usually desired to ensure positive test results are associated with active agents, and not the remains of dead cells. Positive identifications caused by nonliving causative agents may lead to misguided decisions concerning the effectiveness of treatment, and whether patient treatment should be continued or whether the food should be discarded. Consequently, this work was directed toward development of a reverse-transcriptase polymerase chain reaction (RT-PCR)-based in vitro DNA amplification method, which specifically detects only viable cells. Total RNA from both viable and nonviable cells was purified by using a FastPrep Cell Disrupter ([symbol: see text]Bio 101/Savant) and FastRNA extraction reagents ([symbol: see text]Bio 101). The purified RNA was treated with DNase I (RNase-free) to avoid any amplification from the contaminating target DNA. An RT-PCR approach using this rapid and effective method for RNA purification showed amplification of the target mRNA only from the viable cells. The sensitivity of detection of viable cells of V. cholerae was > or = 10(3), which is well within the minimum number of cells (10(5)-10(6)) required for infection. The use of a reliable prokaryotic RNA extraction method followed by RT-PCR amplification of the target mRNA can be used for specific detection of viable microbial pathogen, such as V. cholerae.
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Department of Biology, University of Alabama at Birmingham 35294-1170, USA. BioF003@vabdpo.dpo.uab.edu
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Morgan
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Jones
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Mahbubani
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D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D008722:Methods; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity; D014734:Vibrio cholerae / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012194:RNA-Directed DNA Polymerase