Multiplex PCR detection of clinical and environmental strains ofVibrio vulnificusin shellfish

2004 ◽  
Vol 50 (11) ◽  
pp. 911-922 ◽  
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.

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

2017 ◽  
Vol 61 (4) ◽  
pp. 427-432 ◽  
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.

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

2015 ◽  
Vol 59 (4) ◽  
pp. 489-494 ◽  
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.

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

2018 ◽  
Vol 0 (0) ◽  
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.

scholarly journals Real-Time PCR Detection of Vibrio vulnificus in Oysters: Comparison of Oligonucleotide Primers and Probes Targeting vvhA

2005 ◽  
Vol 71 (10) ◽  
pp. 5702-5709 ◽  
Author(s):  
Gitika Panicker ◽  
Asim K. Bej
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Vulnificus ◽  
Pcr Amplification ◽  
Tissue Homogenate ◽  
Oyster Tissue ◽  
Initial Inoculum ◽  
Oligonucleotide Primers ◽  
Pure Cultures ◽  
Taqman Pcr

ABSTRACT We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene (vvhA) for TaqMan-based real-time PCR method enabling specific detection of Vibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81 V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for all V. vulnificus isolates, also exhibited positive cycle threshold (CT ) values for other Vibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 103 V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification of vvhA. Detection of 3 × 103 CFU V. vulnificus, resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection of V. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe on vvhA for TaqMan-PCR-based detection of V. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducing V. vulnificus-related illnesses and deaths.

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

2018 ◽  
Vol 62 (4) ◽  
pp. 549-554
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 ◽  
Sybr Green ◽  
Sybr Green I ◽  
Chromosomal Marker ◽  
Pcr Products ◽  
Intercalating Dye

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.

scholarly journals Development of ISSR-derived SCAR Marker and SYBR Green I Real-time PCR Method for Detection of Teliospores of Tilletia laevis Kühn

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Zhaoqun Yao ◽  
Dandan Qin ◽  
Delai Chen ◽  
Changzhong Liu ◽  
Wanquan Chen ◽  
...  
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Scar Marker ◽  
Sybr Green ◽  
Sybr Green I ◽  
Tilletia Laevis ◽  
Highly Sensitive ◽  
Pcr Method ◽  
First Time

AbstractCommon bunt, caused by Tilletia laevis Kühn [syn. T. foetida (Wallr) Liro] and Tilletia tritici (Bjerk.) Wint. [syn. T. caries (DC) Tul.], is an important wheat disease worldwide. To quickly differentiate the closely related fungi T. laevis, T. tritici and Tilletia controversa (a pathogen that causes dwarf bunt of wheat and has been requested as a quarantined pathogen in many countries), a rapid diagnostic and detection method for an ISSR molecular marker was developed for the first time in this study. Based on the T. laevis-specific band (1300 bp) amplified by the primer ISSR860, a pair of SCAR primers (L60F/L60R) was designed to amplify a specific 660-bp DNA fragment from the isolates of T. laevis but not other related pathogens. The detection limit of the SCAR marker was 0.4 ng/μl of DNA from T. laevis; moreover, a SYBR Green I real-time PCR method was also successfully developed based on the SCAR marker with the detection limit of 10 fg/μl T. laevis DNA. This is the first report of a rapid, specific and highly sensitive SCAR marker and SYBR Green I real-time PCR method for detection of the teliospores of T. laevis based on ISSR technology. This method allows highly efficient, rapid and accurate differentiation of the pathogen from related pathogens, especially from the very similar pathogens T. tritici and T. controversa.

scholarly journals Rapid Detection of Vibrio vulnificus in Shellfish and Gulf of Mexico Water by Real-Time PCR

2004 ◽  
Vol 70 (1) ◽  
pp. 498-507 ◽  
Author(s):  
Gitika Panicker ◽  
Michael L. Myers ◽  
Asim K. Bej
Keyword(s):  
Gulf Of Mexico ◽  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Vulnificus ◽  
Pcr Amplification ◽  
Tissue Homogenate ◽  
Oyster Tissue ◽  
The Real ◽  
Oligonucleotide Primers ◽  
Number Of Cells

ABSTRACT In this paper we describe optimization of SYBR Green I-based real-time PCR parameters and testing of a large number of microbial species with vvh-specific oligonucleotide primers to establish a rapid, specific, and sensitive method for detection of Vibrio vulnificus in oyster tissue homogenate and Gulf of Mexico water (gulf water). Selected oligonucleotide primers for the vvh gene were tested for PCR amplification of a 205-bp DNA fragment with a melting temperature of approximately 87°C for 84 clinical and environmental strains of V. vulnificus. No amplification was observed with other vibrios or nonvibrio strains with these primers. The minimum level of detection by the real-time PCR method was 1 pg of purified genomic DNA or 102 V. vulnificus cells in 1 g of unenriched oyster tissue homogenate or 10 ml of gulf water. It was possible to improve the level of detection to one V. vulnificus cell in samples that were enriched for 5 h. The standard curves prepared from the real-time PCR cycle threshold values revealed that there was a strong correlation between the number of cells in unenriched samples and the number of cells in enriched samples. Detection of a single cell of V. vulnificus in 1 g of enriched oyster tissue homogenate is in compliance with the recent Interstate Shellfish Sanitation Conference guidelines. The entire detection method, including sample processing, enrichment, and real-time PCR amplification, was completed within 8 h, making it a rapid single-day assay. Rapid and sensitive detection of V. vulnificus would ensure a steady supply of postharvest treated oysters to consumers, which should help decrease the number of illnesses or outbreaks caused by this pathogen.

scholarly journals An Optimized Real-Time PCR to Avoid Species-/Tissue-Associated Inhibition for H5N1 Detection in Ferret and Monkey Tissues

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
LingJun Zhan ◽  
LinLin Bao ◽  
FengDi Li ◽  
Qi Lv ◽  
LiLi Xu ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rna Isolation ◽  
High Specificity ◽  
Sybr Green ◽  
Pcr Inhibitors ◽  
Specificity And Sensitivity ◽  
Avian Influenza Virus H5n1 ◽  
Pcr Method ◽  
Tissue Specimens

The real-time PCR diagnostics for avian influenza virus H5N1 in tissue specimens are often suboptimal, since naturally occurring PCR inhibitors present in samples, or unanticipated match of primer to unsequenced species’ genome. With the principal aim of optimizing the SYBR Green real-time PCR method for detecting H5N1 in ferret and monkey (Chinese rhesus macaque) tissue specimens, we screened various H5N1 gene-specific primer pairs and tested their ability to sensitively and specifically detect H5N1 transcripts in the infected animal tissues, then we assessed RNA yield and quality by comparing Ct values obtained from the standard Trizol method, and four commonly used RNA isolation kits with small modifications, including Roche High Pure, Ambion RNAqueous, BioMIGA EZgene, and Qiagen RNeasy. The results indicated that a single primer pair exhibited high specificity and sensitivity for H5N1 transcripts in ferret and monkey tissues. Each of the four kits and Trizol reagent produced high-quality RNA and removed all or nearly all PCR inhibitors. No statistically significant differences were found between the Ct values from the isolation methods. So the optimized SYBR Green real-time PCR could avoid species- or tissue-associated PCR inhibition in detecting H5N1 in ferret and monkey tissues, including lung and small intestine.

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