Retrospective study of a plague outbreak by multiplex-PCR

Melo, Amanda Campelo Lima de; Almeida, Alzira Maria Paiva de; Leal, Nilma Cintra

doi:10.1046/j.1472-765x.2003.01377.x

Cited by 15 publications

(9 citation statements)

References 17 publications

(35 reference statements)

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“…Therefore, numerous rapid tests for the detection of Y. pestis have been developed. Most of these include different variants of polymerase chain reaction, PCR [9]–[15]. Disadvantages of conventional PCR tests include the need to analyze the PCR products by gel electrophoresis and frequent contamination of the laboratory by the amplicons.…”

Section: Introductionmentioning

confidence: 99%

Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

Sergueev

Borschel

et al. 2010

PLoS ONE

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Background Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics.Methodology/Principal FindingsThe objective of this work was to develop an alternative to conventional phage lysis tests – a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages ϕA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. ϕA1122-specific qPCR enabled the detection of an initial bacterial concentration of 103 CFU/ml (equivalent to as few as one Y. pestis cell per 1-µl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, ϕA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR.Conclusions/SignificanceThus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria.

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Section: Introductionmentioning

confidence: 99%

Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

Sergueev

Borschel

et al. 2010

PLoS ONE

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“…These techniques are particularly useful when causative microorganisms are resistant to cultivation or in retrospective studies of samples in which microorganisms are no longer viable 10,27–29. This study was designed to compare the frequency of bacterial infection between the amniotic cavity and the chorioamniotic membranes during MIAC and to determine if bacteria normally colonize the intrauterine compartment during pregnancy.…”

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confidence: 99%

Widespread microbial invasion of the chorioamniotic membranes is a consequence and not a cause of intra-amniotic infection

Kim

Romero

Gervasi

et al. 2009

Laboratory Investigation

133

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“…Y. pestis strains isolated from plague patients usually contain all 3 virulence plasmids, but these may be lacking in atypical strains; therefore, molecular detection strategies usually include targets on each plasmid. Recent standard PCR methods (7)(8)(9)(10)(11) and real-time PCR assays (12)(13)(14)(15) have primarily used specific virulence gene targets encoded on these 3 plasmids. However, in the opinion of Chain et al (16 ), "the presence of these plasmids by themselves cannot account for the remarkable increase in virulence observed in Y. pestis".…”

mentioning

confidence: 99%

Real-Time PCR Assays Targeting a Unique Chromosomal Sequence of Yersinia pestis

et al. 2005

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Background: Yersinia pestis, the causative agent of the zoonotic infection plague, is a major concern as a potential bioweapon. Current real-time PCR assays used for Y. pestis detection are based on plasmid targets, some of which may generate false-positive results. Methods: Using the yp48 gene of Y. pestis, we designed and tested 2 real-time TaqMan ® minor groove binder (MGB) assays that allowed us to use chromosomal genes as both confirmatory and differential targets for Y. pestis. We also designed several additional assays using both Simple-Probe

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Retrospective study of a plague outbreak by multiplex-PCR

Cited by 15 publications

References 17 publications

Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

Widespread microbial invasion of the chorioamniotic membranes is a consequence and not a cause of intra-amniotic infection

Real-Time PCR Assays Targeting a Unique Chromosomal Sequence of Yersinia pestis

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