SYBR®Green qPCR Salmonella detection system allowing discrimination at the genus, species and subspecies levels

Barbau-Piednoir, Elodie; Bertrand, Sophie; Mahillon, Jacques; Roosens, Nancy; Botteldoorn, Nadine

doi:10.1007/s00253-013-5234-x

Cited by 33 publications

(24 citation statements)

References 47 publications

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“…The selectivity test was composed of two steps as previously reported for the validation of qPCR methods for food pathogens (Barbau-Piednoir et al 2013a , b ). Firstly, a preliminary selectivity test was carried out on the target species ( A. versicolor IHEM 18884) and two nontarget species (i.e., A. fumigatus IHEM 3562 and P. chrysogenum IHEM 20859), using 15,000 theoretical copies of gDNA.…”

Section: Methodsmentioning

confidence: 99%

Development and performance assessment of a qualitative SYBR® green real-time PCR assay for the detection of Aspergillus versicolor in indoor air

Libert

Chasseur

Bladt³

et al. 2015

Appl Microbiol Biotechnol

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Currently, contamination of indoor environment by fungi and molds is considered as a public health problem. The monitoring of indoor airborne fungal contamination is a common tool to help understanding the link between fungi in houses and respiratory problems. Classical analytical monitoring methods, based on cultivation and microscopic identification, depend on the growth of the fungi. Consequently, they are biased by difficulties to grow some species on certain culture media and under certain conditions or by noncultivable or dead fungi that can consequently not be identified. However, they could have an impact on human health as they might be allergenic. Since molecular methods do not require a culture step, they seem an excellent alternative for the monitoring of indoor fungal contaminations. As a case study, we developed a SYBR® green real-time PCR-based assay for the specific detection and identification of Aspergillus versicolor, which is frequently observed in indoor environment and known to be allergenic. The developed primers amplify a short region of the internal transcribed spacer 1 from the 18S ribosomal DNA complex. Subsequently, the performance of this quantitative polymerase chain reaction (qPCR) method was assessed using specific criteria, including an evaluation of the selectivity, PCR efficiency, dynamic range, and repeatability. The limit of detection was determined to be 1 or 2 copies of genomic DNA of A. versicolor. In order to demonstrate that this SYBR® green qPCR assay is a valuable alternative for monitoring indoor fungal contamination with A. versicolor, environmental samples collected in contaminated houses were analyzed and the results were compared to the ones obtained with the traditional methods.

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Section: Methodsmentioning

confidence: 99%

Development and performance assessment of a qualitative SYBR® green real-time PCR assay for the detection of Aspergillus versicolor in indoor air

Libert

Chasseur

Bladt³

et al. 2015

Appl Microbiol Biotechnol

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“…Consequently, this system forms an efficient and cost-/time-saving tool for the detection of authorised and unauthorised GMO in food and feed samples. This type of screening tools can be applied in other GMO detection laboratories but can also be adapted to other scientific fields such as the detection of food-borne pathogens [50,51]. Additionally, these methods can serve as a basis to develop other GMO detection tools using new technologies such as xMAP [52][53][54].…”

Section: Resultsmentioning

confidence: 99%

New qualitative trait-specific SYBR®Green qPCR methods to expand the panel of GMO screening methods used in the CoSYPS

Broeders

Fraiture

Vandermassen

et al. 2015

Eur Food Res Technol

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specificity and repeatability were assessed as well as the robustness of the methods. Additionally, the reproducibility was evaluated by transferring the methods to a second laboratory. Both methods allow a specific, sensitive and repeatable detection of the respective targets in food and feed samples and can be easily applied in a routine laboratory. Moreover, they can be used together with previously validated SYBR ® Green qPCR methods to expand the panel of screening methods. This allows an extended coverage of the GM events authorised in the EU and adds discriminative power to the screening phase.

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“…This method does not require the design of a separate probe, such as with the TaqMan qPCR technique, which can be complicated and expensive. Moreover, SYBR green qPCR appears to be more sensitive than the TaqMan probe-based qPCR [29,36]. The speci city of SYBR-green qPCR may also provide information regarding the amount of DNA ampli cation by using the melting curve analysis [37].…”

Section: Discussionmentioning

confidence: 99%

“…SYBR green qPCR has recently been used in many studies to detect, discriminate, and quantify species as, for example, in the discrimination and quanti cation of Leishmania in human samples and the discrimination of species and subspecies of Salmonella. The technique was also employed for the detection of Opisthorchis viverrini and Haplorchis taichui from human stool samples [28][29][30].…”

Section: Introductionmentioning

confidence: 99%

SYBR-Green-Based Quantitative Real-Time PCR for Discriminating Between Closely Related Angiostrongylus Cantonensis and A. Malaysisensis

Jakkul¹,

Chaisiri²,

Saralamba³

et al. 2020

Preprint

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Background: Angiostrongylus cantonensis is a well-known pathogen causing human angiostrongyliasis eosinophilic meningitis. Humans, as accidental hosts, are infected by eating undercooked snails containing third-stage larvae. A. malaysiensis is closely related to A. cantonensis and has been described as a potential human pathogen. Recently, the two species have been reported to have overlapping distributions in the same endemic area, particularly in the Indochina region. Because of their similar morphological characteristics, misidentification often occurs, particularly of the third-stage larva in the snail intermediate host. Methods: We designed species-specific primers to mitochondrial cytochrome b, which was used as a genetic marker. SYBR-green quantitative real-time PCR (qPCR) was employed to quantitatively detect and identify the third-stage larvae and tissue debris in the cerebrospinal fluid (CSF) of a patient, and to quantify third-stage larvae in the snail Achatina fulica collected from the field.Results: The newly designed primers were highly specific and sensitive, even when using conventional PCR. SYBR green qPCR quantitatively detected around 10−4 ng of genomic DNA from one larva and facilitated the specific detection and identification of parasitic genetic material from the CSF of a patient with angiostrongyliasis. The method also estimated the number of larvae in A. fulica and revealed that the primary source of Angiostrongylus infection in the King Rama IX public park study area was A. malaysiensis; although, the two Angiostrongylus species each infected 10% of the snails. Conclusions: Our SYBR green qPCR method is a useful and inexpensive technique for parasite identification and has sufficient sensitivity and specificity to detect a single larva and simultaneously discriminate between A. cantonensis and A. malaysiensis. The number of larvae infecting or co-infecting the snail intermediate host can also be estimated. In future research, this qPCR method could be employed in a molecular survey of A. cantonensis and A. malaysiensis occurrence within intermediate and definitive hosts. The technique should also be applied in a study analyzing CSF specimens from patients with eosinophilic meningitis to assess the usefulness of the method for clinical diagnosis.

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SYBR®Green qPCR Salmonella detection system allowing discrimination at the genus, species and subspecies levels

Cited by 33 publications

References 47 publications

Development and performance assessment of a qualitative SYBR® green real-time PCR assay for the detection of Aspergillus versicolor in indoor air

Development and performance assessment of a qualitative SYBR® green real-time PCR assay for the detection of Aspergillus versicolor in indoor air

New qualitative trait-specific SYBR®Green qPCR methods to expand the panel of GMO screening methods used in the CoSYPS

SYBR-Green-Based Quantitative Real-Time PCR for Discriminating Between Closely Related Angiostrongylus Cantonensis and A. Malaysisensis

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