Evaluation of real-time PCR based on SYBR Green I fluorescent dye for detection of Bacillus anthracis strains in biological samples

AbstractIntroduction:The aim of the study was the application and evaluation of real-time PCRs based on the fluorescence of SYBR Green I intercalating dye for the detection of threeBacillus anthracisgenes in contaminated liver and blood samples. The goals for detection wererpoBgene as a chromosomal marker,paggene located on plasmid pXO1, andcapCgene located on plasmid pXO2.Material and Methods:FiveB. anthracisstrains were used for the experiments. Additionally, single strains of other species of the genusBacillus,i.e.B. cereus,B. brevis,B. subtilis, andB. megaterium, and strains of six other species were used for evaluation of the specificity of the tests. Three SYBR Green I real-time PCRs were conducted allowing confirmation ofB. anthracisin the biological samples.Results:The observation of amplification curves in real-time PCRs enabled the detection of the chromosomally encodedrpoBgene,paggene, andcapCgene ofB. anthracis. The specificity of the tests was confirmed by estimation of the melting temperature of the PCR products. The sensitivity and linearity of the reactions were determined using regression coefficients. Strains of other microbial species did not reveal real-time PCR products.Conclusion:All real-time PCRs for the detection ofB. anthracisin biological samples demonstrated a significant sensitivity and high specificity.

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Evaluation of Real-Time Pcr Based on Sybr Green I Fluorescent Dye for Detection of Bacillus Anthracis Strains in Biological Samples

Journal of Veterinary Research ◽

10.2478/jvetres-2019-0001 ◽

2018 ◽

Vol 0 (0) ◽

Cited By ~ 1

Author(s):

Agnieszka Kędrak-Jabłońska ◽

Sylwia Budniak ◽

Anna Szczawińska ◽

Monika Reksa ◽

Marek Krupa ◽

...

Keyword(s):

Bacillus Anthracis ◽

Real Time ◽

Real Time Pcr ◽

Biological Samples ◽

High Specificity ◽

Rpob Gene ◽

Sybr Green ◽

Sybr Green I ◽

Chromosomal Marker ◽

Pcr Products

AbstractIntroduction: The aim of the study was the application and evaluation of real-time PCRs based on the fluorescence of SYBR Green I intercalating dye for the detection of three Bacillus anthracis genes in contaminated liver and blood samples. The goals for detection were rpoB gene as a chromosomal marker, pag gene located on plasmid pXO1, and capC gene located on plasmid pXO2. Material and Methods: Five B. anthracis strains were used for the experiments. Additionally, single strains of other species of the genus Bacillus, i.e. B. cereus, B. brevis, B. subtilis, and B. megaterium, and strains of six other species were used for evaluation of the specificity of the tests. Three SYBR Green I real-time PCRs were conducted allowing confirmation of B. anthracis in the biological samples. Results: The observation of amplification curves in real-time PCRs enabled the detection of the chromosomally encoded rpoB gene, pag gene, and capC gene of B. anthracis. The specificity of the tests was confirmed by estimation of the melting temperature of the PCR products. The sensitivity and linearity of the reactions were determined using regression coefficients. Strains of other microbial species did not reveal real-time PCR products. Conclusion: All real-time PCRs for the detection of B. anthracis in biological samples demonstrated a significant sensitivity and high specificity.

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Detection of Listeria spp. and Listeria monocytogenes in biological samples by SYBR Green I and TaqMan probe-based real-time PCRs

Journal of Veterinary Research ◽

10.1515/jvetres-2017-0069 ◽

2017 ◽

Vol 61 (4) ◽

pp. 427-432 ◽

Cited By ~ 2

Author(s):

Agnieszka Kędrak-Jabłońska ◽

Sylwia Budniak ◽

Marek Krupa ◽

Anna Szczawińska ◽

Monika Reksa ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Biological Samples ◽

High Specificity ◽

Sybr Green ◽

Sybr Green I ◽

Taqman Probe ◽

Rdna Gene ◽

Intercalating Dye

AbstractIntroduction:The aim of the study was the application and comparison of real-time PCR methods based on the fluorescence of SYBR Green I intercalating dye and TaqMan probes for the detection of the 23S rDNA gene ofListeriaspp. and thehlyA gene ofListeria monocytogenesin biological samples of the liver, brain, and blood.Material and Methods:Five strains ofL. monocytogenesand single strains of each speciesL. ivanovii,L. innocua,L. grayi,L. welshimeri,andL. seeligeriwere used for the experiments. Additionally, five strains of other species of bacteria were used for evaluation of the specificity of tests. In the first stage of the study SYBR Green I real-time PCRs, one allowing detection of the 23S rDNA gene and two based on the amplification thehlyA gene, were performed. In the next part, three TaqMan probe-based real-time PCRs allowing confirmation of belonging toListeriaspp. andL. monocytogeneswere conducted.Results:The observation of amplification curves in real-time PCRs enabled the detection of both genes. A high regression coefficient of 0.99 was found for all reactions. Specific amplification products were obtained for the 23S rDNA andhlyA genes which confirm their belonging toListeriaspp. andL. monocytogenes, respectively. Other microbial species did not reveal real-time PCR products.Conclusion:Both real-time PCR methods for the detection ofListeriaspp. andL. monocytogenesin biological samples demonstrated a significant sensitivity and high specificity.

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Application of SYBR Green I and TaqMan probe-based real-time PCRs for the identification of Listeria spp. and Listeria monocytogenes

Bulletin of the Veterinary Institute in Pulawy ◽

10.1515/bvip-2015-0073 ◽

2015 ◽

Vol 59 (4) ◽

pp. 489-494 ◽

Cited By ~ 2

Author(s):

Agnieszka Kędrak-Jabłońska ◽

Sylwia Budniak ◽

Anna Szczawińska ◽

Monika Reksa ◽

Marek Krupa ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

High Specificity ◽

Sybr Green ◽

Sybr Green I ◽

Taqman Probe ◽

Rdna Gene ◽

Intercalating Dye ◽

Hlya Gene

Abstract The aim of the study was the application and comparison of real-time PCR methods based on the fluorescence of SYBR Green I intercalating dye and TaqMan probes for the detection of the 23S rDNA gene of Listeria spp. and the hlyA gene of Listeria monocytogenes. Five strains of L. monocytogenes and single strains of each of the species: L. ivanovii, L. innocua, L. grayi, L. welshimeri, and L. seeligeri were used for the experiments. Additionally, five strains of other species of bacteria were used for evaluation of the specificity of the tests. QuantiTect SYBR Green PCR and QuantiTect Probe PCR kits were selected for the study. In the first stage of the study, SYBR Green I real-time PCRs were performed under several methods, the first one allowing detection of the 23S rDNA gene and the remainder based on the amplification of the hlyA gene. In the next part, three varied in method TaqMan probe-based real-time PCRs allowing confirmation of strains belonging to Listeria spp. and L. monocytogenes were conducted. The observation of amplification curves in real-time PCR methods enabled the detection of both genes, and these methods demonstrated a significant sensitivity and high specificity. A high regression coefficient of 0.99 was found for all reactions. Specific amplification products were obtained for the 23S rDNA and hlyA genes, which confirmed the tested strains as Listeria spp. and L. monocytogenes respectively. Isolates of other microbial species did not yield real-time PCR products.

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Multiplex PCR detection of clinical and environmental strains ofVibrio vulnificusin shellfish

Canadian Journal of Microbiology ◽

10.1139/w04-085 ◽

2004 ◽

Vol 50 (11) ◽

pp. 911-922 ◽

Cited By ~ 34

Author(s):

Gitika Panicker ◽

Michael C.L Vickery ◽

Asim K Bej

Keyword(s):

Multiplex Pcr ◽

Real Time ◽

Real Time Pcr ◽

Vibrio Vulnificus ◽

High Specificity ◽

Sybr Green ◽

Sybr Green I ◽

Tissue Homogenate ◽

Oyster Tissue ◽

Pcr Method

In this study, we developed a PCR-based rapid detection method for clinically important pathogenic strains of Vibrio vulnificus. Positive amplification of the 504-bp viuB fragment was seen in all 22 clinical isolates tested but only in 8 out of 33 environmental isolates. The combination of the species-specific 205-bp vvh fragment along with viuB in a multiplexed PCR enabled us to confirm the presence of potentially pathogenic strains of V. vulnificus. No amplification of other Vibrio spp. or non-Vibrio bacteria was evidenced, suggesting a high specificity of detection by this method. The sensitivity of detection for both targeted genes was 10 pg of purified DNA, which correlated with 103V. vulnificus CFU in 1 mL of pure culture or 1 g un-enriched seeded oyster tissue homogenate. This sensitivity was improved to 1 CFU per gram of oyster tissue homogenate in overnight-enriched samples. A SYBR Green I based real-time PCR method was also developed that was shown to produce results consistent with the conventional PCR method. Application of the multiplexed real-time PCR to natural oyster tissue homogenates exhibited positive detection of vvh in 51% of the samples collected primarily during the summer months; however, only 15% of vvh positive samples exhibited viuB amplicons. The rapid, sensitive, and specific detection of clinically important pathogenic V. vulnificus in shellfish would be beneficial in reducing illnesses and deaths caused by this pathogen.Key words: Vibrio, multiplex PCR, shellfish, SYBR Green I, real-time PCR.

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