Rapid detection of virulence stx2 gene of Enterohemorrhagic Escherichia coli using two-step ultra-rapid real-time PCR

Kim, Il-Wook; Kang, Minhee; Kwon, Soon-Hwan; Cho, Seung-Hak; Yoo, Byueng–Su; Han, Sanghoon; Yoon, Byoung-Su

doi:10.1007/s10529-010-0205-0

Cited by 6 publications

(3 citation statements)

References 20 publications

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“…The DNA template was extracted from a 1-mL solution of Listeria monocytogenes via the boiling method and amplified by PCR. The IAC preparation involved two PCR amplifications [25]. The first step of PCR amplification employed a total reaction volume of 25 μL: 1 μL of DNA template,2 μL dNTP, 2.5 μL 10x PCR buffer (Mg 2+ ), 1 μL of the upstream (IAC-F, 10 μmol/L) heterozygous primers, 1 μL of downstream (IAC-R, 10μmol/L) heterozygous primers.…”

Section: Methodsmentioning

confidence: 99%

Contrast of Real-Time Fluorescent PCR Methods for Detection of Escherichia coli O157:H7 and of Introducing an Internal Amplification Control

2019

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Various constituents in food specimens can inhibit the PCR assay and lead to false-negative results. An internal amplification control was employed to monitor the presence of false-negative results in PCR amplification. In this study, the objectives were to compare the real-time PCR-based method by introducing a competitive internal amplification control (IAC) for the detection of Escherichia O157:H7 with respect to the specificity of the primers and probes, analytical sensitivity, and detection limits of contamination-simulated drinking water. Additionally, we optimized the real-time fluorescent PCR detection system for E. coli O157:H7. The specificity of primers and probes designed for the rfbE gene was evaluated using four kinds of bacterial strains, including E. coli O157:H7, Staphylococcus aureus, Salmonella and Listeria monocytogenes strains. The real time PCR assay unambiguously distinguished the E. coli O157:H7 strains after 16 cycles. Simultaneously, the lowest detection limit for E. coli O157:H7 in water samples introducing the IAC was 104 CFU/mL. The analytical sensitivity in water samples had no influence on the detection limit compared with that of pure cultures. The inclusion of an internal amplification control in the real-time PCR assay presented a positive IAC amplification signal in artificially simulated water samples. These results indicated that real-time fluorescent PCR combined with the IAC possessed good characteristics of stability, sensitivity, and specificity. Consequently, the adjusted methods have the potential to support the fast and sensitive detection of E. coli O157:H7, enabling accurate quantification and preventing false negative results in E. coli O157:H7 contaminated samples.

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Section: Methodsmentioning

confidence: 99%

Contrast of Real-Time Fluorescent PCR Methods for Detection of Escherichia coli O157:H7 and of Introducing an Internal Amplification Control

2019

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“…The first step consisted of identifying genes of interest, either genus or pathotype specific, by means of a bibliographic study (Clements et al 2012 ; Croxen and Finlay 2010 ). The second step included the collection of primer sequences available in the literature targeting the selected genes giving an amplicon between 60 and 120 bp (Anklam et al 2012 ; Botteldoorn et al 2003 ; Fukushima et al 2009 ; Kim et al 2010 ; Nielsen and Andersen 2003 ; Pavlovic et al 2010 ; Perelle et al 2004 ; Sharma et al 1999 ; Takahashi et al 2009 ; Thiem et al 2004 ; Tzschoppe et al 2012 ). If none were found, primer pairs were designed, preferentially within conserved regions, using the “Primer 3” program ( http://frodo.wi.mit.edu/primer3/ (Rozen and Skaletsky 2000 )) with the “product size range” specification set at “60 to 120 bp” and “primer size” optimal set at “22 bases.” In the third step, a collection of bacterial DNA sequences of other foodborne pathogenic bacteria and bacteria naturally present in food matrices was retrieved from the NCBI public database ( http://www.ncbi.nlm.nih.gov/sites/entrez ).…”

Section: Methodsmentioning

confidence: 99%

Detection and discrimination of five E. coli pathotypes using a combinatory SYBR® Green qPCR screening system

Barbau-Piednoir

Denayer

Botteldoorn

et al. 2018

Appl Microbiol Biotechnol

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A detection and discrimination system for five Escherichia coli pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic E. coli (CoSYPS Path E. coli). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of E. coli: shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) E. coli. The SYBR® Green qPCR assays target the uidA, ipaH, eae, aggR, aaiC, stx1, and stx2 genes. uidA controls for E. coli presence and all the other genes are specific targets of E. coli pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs’ design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path E. coli system was subsequently evaluated on four food matrices artificially contaminated with pathogenic E. coli. It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.Electronic supplementary materialThe online version of this article (10.1007/s00253-018-8820-0) contains supplementary material, which is available to authorized users.

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“…In recent years, micro‐scale PCR chips have been developed, which can supply high‐speed temperature homogeneity inside the reaction tubes. Moreover, a real‐time micro‐scale chip‐based PCR system has been developed, and this system operates with high specificity and sensitivity: 40 thermal cycles in less than 20 Min . However, the general weakness of the PCR‐based detection method is reliability, and that is concern whether an exact specific gene is amplified.…”

Section: Introductionmentioning

confidence: 99%

Application of ultra‐rapid qPCR and DNA chips for viral RNA detection and confirmation

Kim

Cho

Yoon

2018

Biotech and App Biochem

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The rapid and accurate detection of the presence of microorganisms, such as viruses, has been an important issue in the fields of public health, as well as agriculture. A PCR-based detection method has been developed and applied in these fields to determine the presence of specific pathogens. Although the major advantage of real-time PCR is the monitoring of amplification and ability to quantify the template genes, the method described here should solve the problem of nonspecific product synthesis. We obtained viral RNA from infected samples by freezing and thawing; we rapidly synthesized cDNA from RNA, and then amplified the cDNA by rapid PCR in 10 Min. Finally, the PCR products were hybridized and quickly confirmed to be the target analyte on a DNA chip. Our newly proposed methods overcome the drawbacks of PCR-based detection and provide three additional advantages, namely, rapidly obtaining large amounts of RNA from samples, quickly detecting infective or pathogenic genes, and speedily confirming the detected exogenous genes. This application might be useful for detecting viral RNA and for the diagnosis of RNA virus-mediated diseases.

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Rapid detection of virulence stx2 gene of Enterohemorrhagic Escherichia coli using two-step ultra-rapid real-time PCR

Cited by 6 publications

References 20 publications

Contrast of Real-Time Fluorescent PCR Methods for Detection of Escherichia coli O157:H7 and of Introducing an Internal Amplification Control

Contrast of Real-Time Fluorescent PCR Methods for Detection of Escherichia coli O157:H7 and of Introducing an Internal Amplification Control

Detection and discrimination of five E. coli pathotypes using a combinatory SYBR® Green qPCR screening system

Application of ultra‐rapid qPCR and DNA chips for viral RNA detection and confirmation

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