scholarly journals New Triplex Real-Time PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Feces

2008 ◽  
Vol 74 (9) ◽  
pp. 2751-2758 ◽  
Author(s):  
H. Schönenbrücher ◽  
A. Abdulmawjood ◽  
K. Failing ◽  
M. Bülte
Keyword(s):  
Real Time ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Detection System ◽  
Locked Nucleic Acid ◽  
Amplification Efficiency ◽  
Bacterial Strains ◽  
Fecal Samples ◽  
Bovine Feces

ABSTRACT In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2 sequences of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105 M. avium subsp. paratuberculosis strains of bovine, ovine, and human origin and 100 non-M. avium subsp. paratuberculosis strains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogram M. avium subsp. paratuberculosis DNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection of M. avium subsp. paratuberculosis DNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqManmgb and locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection of M. avium subsp. paratuberculosis from bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 102 CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknown M. avium subsp. paratuberculosis status resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method.

scholarly journals Validation of a Novel Diagnostic Approach Combining the VersaTREK™ System for Recovery and Real-Time PCR for the Identification of Mycobacterium chimaera in Water Samples

2021 ◽  
Vol 9 (5) ◽  
pp. 1031
Author(s):  
Roberto Zoccola ◽  
Alessia Di Blasio ◽  
Tiziana Bossotto ◽  
Angela Pontei ◽  
Maria Angelillo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Detection System ◽  
Bacterial Load ◽  
Microbial Control ◽  
Hospital Setting ◽  
Sample Volume ◽  
Analytical Sensitivity ◽  
Initial Water

Mycobacterium chimaera is an emerging pathogen associated with endocarditis and vasculitis following cardiac surgery. Although it can take up to 6–8 weeks to culture on selective solid media, culture-based detection remains the gold standard for diagnosis, so more rapid methods are urgently needed. For the present study, we processed environmental M. chimaera infected simulates at volumes defined in international guidelines. Each preparation underwent real-time PCR; inoculates were placed in a VersaTREK™ automated microbial detection system and onto selective Middlebrook 7H11 agar plates. The validation tests showed that real-time PCR detected DNA up to a concentration of 10 ng/µL. A comparison of the isolation tests showed that the PCR method detected DNA in a dilution of ×102 CFU/mL in the bacterial suspensions, whereas the limit of detection in the VersaTREK™ was <10 CFU/mL. Within less than 3 days, the VersaTREK™ detected an initial bacterial load of 100 CFU. The detection limit did not seem to be influenced by NaOH decontamination or the initial water sample volume; analytical sensitivity was 1.5 × 102 CFU/mL; positivity was determined in under 15 days. VersaTREK™ can expedite mycobacterial growth in a culture. When combined with PCR, it can increase the overall recovery of mycobacteria in environmental samples, making it potentially applicable for microbial control in the hospital setting and also in environments with low levels of contamination by viable mycobacteria.

scholarly journals Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Ellen Kathrin Link ◽  
Matthias Eddicks ◽  
Liangliang Nan ◽  
Mathias Ritzmann ◽  
Gerd Sutter ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Diagnostic Sensitivity ◽  
Locked Nucleic Acid ◽  
Porcine Circovirus ◽  
Amplification Efficiency ◽  
Pcr Assays

Abstract Background The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR. Methods Based on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes. Results All eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g. Conclusion Genotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers.

scholarly journals Development of an F57 Sequence-Based Real-Time PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Milk

2005 ◽  
Vol 71 (10) ◽  
pp. 5957-5968 ◽  
Author(s):  
T. Tasara ◽  
R. Stephan
Keyword(s):  
Real Time ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Quantitative Estimation ◽  
Bacterial Species ◽  
Detection Sensitivity ◽  
Bacterial Strains ◽  
Pcr Assay ◽  
Sequence Element ◽  
Milk Samples

ABSTRACT A light cycler-based real-time PCR (LC-PCR) assay that amplifies the F57 sequence of Mycobacterium avium subsp. paratuberculosis was developed. This assay also includes an internal amplification control template to monitor the amplification conditions in each reaction. The targeted F57 sequence element is unique for M.avium subsp. paratuberculosis and is not known to exist in any other bacterial species. The assay specificity was demonstrated by evaluation of 10 known M. avium subsp. paratuberculosis isolates and 33 other bacterial strains. The LC-PCR assay has a broad linear range (2 × 101 to 2 ×106 copies) for quantitative estimation of the number of M. avium subsp. paratuberculosis F57 target copies in positive samples. To maximize the assay's detection sensitivity, an efficient strategy for isolation of M. avium subsp. paratuberculosis DNA from spiked milk samples was also developed. The integrated procedure combining optimal M. avium subsp. paratuberculosis DNA isolation and real-time PCR detection had a reproducible detection limit of about 10 M. avium subsp. paratuberculosis cells per ml when a starting sample volume of 10 ml of M. avium subsp. paratuberculosis-spiked milk was analyzed. The entire process can be completed within a single working day and is suitable for routine monitoring of milk samples for M. avium subsp. paratuberculosis contamination. The applicability of this protocol for naturally contaminated milk was also demonstrated using milk samples from symptomatic M. avium subsp. paratuberculosis-infected cows, as well as pooled samples from a dairy herd with a confirmed history of paratuberculosis.

scholarly journals High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides.

2021 ◽  
Author(s):  
Sarah Leah Downs ◽  
Shabir Ahmed Madhi ◽  
Lara van Der Merwe ◽  
Marta Coelho Nunes ◽  
Courtney Paige Olwagen
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Locked Nucleic Acid ◽  
Clinical Samples ◽  
Taqman Assay ◽  
Vaccine Serotypes ◽  
Serotype Replacement ◽  
The Impact

Abstract Current real-time Polymerase Chain Reaction (qPCR) methods are unable to distinguish serotypes 6A from 6B, 18C from 18A/B and 22F from 22A. We established a nanofluidic real-time PCR (Fluidigm) for serotyping that included Dual-Priming-Oligonucleotides (DPO), a Locked-Nucleic-Acid (LNA) probe and TaqMan assay-sets for high-throughput serotyping. The designed assay-sets target capsular gene wciP in serogroup 6, wciX and wxcM in serogroup 18, and wcwA in serogroup 22. An algorithm combining results from published assay-sets (6A/B/C/D; 6C/D; 18A/B/C; 22A/F) and designed assay-sets for 6A/C; 18B/C/F; 18C/F, 18F and 22F was validated through blind analysis of 1973 archived clinical samples collected from South African children ≤ 5-years-old (2009-11), previously serotyped with the culture-based Quellung method. All assay-sets were efficient (92–101%), had low variation between replicates (R2 > 0.98), and were able to detect targets at a limit of detection (LOD) of < 100 Colony-Forming-Units (CFU)/ml of sample. There was high concordance (Kappa = 0.73–0.92); sensitivity (85–100%) and specificity (96–100%) for Fluidigm compared with Quellung for serotyping 6A; 6B; 6C; 18C and 22F. Fluidigm distinguishes vaccine-serotypes 6A, 6B, 18C, next-generation PCV-serotype 22F and non-vaccine-serotypes 6C, 6D, 18A, 18B, 18F and 22A. Discriminating single serotypes is important for assessing serotype replacement and the impact of PCVs on vaccine- and non-vaccine serotypes.

scholarly journals High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
S. L. Downs ◽  
S. A. Madhi ◽  
L. Van der Merwe ◽  
M. C. Nunes ◽  
C. P. Olwagen
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Locked Nucleic Acid ◽  
Clinical Samples ◽  
Taqman Assay ◽  
Vaccine Serotypes ◽  
Serotype Replacement ◽  
The Impact

AbstractCurrent real-time high-throughput Polymerase Chain Reaction (qPCR) methods do not distinguish serotypes 6A from 6B, 18C from 18A/B and 22F from 22A. We established a nanofluidic real-time PCR (Fluidigm) for serotyping that included Dual-Priming-Oligonucleotides (DPO), a Locked-Nucleic-Acid (LNA) probe and TaqMan assay-sets for high-throughput serotyping. The designed assay-sets target capsular gene wciP in serogroup 6, wciX and wxcM in serogroup 18, and wcwA in serogroup 22. An algorithm combining results from published assay-sets (6A/B/C/D; 6C/D; 18A/B/C; 22A/F) and designed assay-sets for 6A/C; 18B/C/F; 18C/F, 18F and 22F was validated through blind analysis of 1973 archived clinical samples collected from South African children ≤ 5-years-old (2009–2011), previously serotyped with the culture-based Quellung method. All assay-sets were efficient (92–101%), had low variation between replicates (R2 > 0.98), and were able to detect targets at a limit of detection (LOD) of < 100 Colony-Forming-Units (CFU)/mL of sample. There was high concordance (Kappa = 0.73–0.92); sensitivity (85–100%) and specificity (96–100%) for Fluidigm compared with Quellung for serotyping 6A; 6B; 6C; 18C and 22F. Fluidigm distinguishes vaccine-serotypes 6A, 6B, 18C, next-generation PCV-serotype 22F and non-vaccine-serotypes 6C, 6D, 18A, 18B, 18F and 22A. Discriminating single serotypes is important for assessing serotype replacement and the impact of PCVs on vaccine- and non-vaccine serotypes.

scholarly journals Locked Nucleic Acid Hydrolysis Probes for the Specific Identification of Probiotic Strains Bifidobacterium animalis subsp. lactis DSM 15954 and Bi-07™

2021 ◽  
Vol 12 ◽  
Author(s):  
Hanan R. Shehata ◽  
Anthony Kiefer ◽  
Wesley Morovic ◽  
Steven G. Newmaster
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Probiotic Strain ◽  
Locked Nucleic Acid ◽  
Identification Methods ◽  
Bifidobacterium Animalis ◽  
Pcr Assays ◽  
Pcr Targeting

Probiotic health benefits are now well-recognized to be strain specific. Probiotic strain characterization and identification is thus important in clinical research and in the probiotic industry. This is becoming especially important with reports of probiotic products failing to meet the declared strain content, potentially compromising their efficacy. Availability of reliable identification methods is essential for strain authentication during discovery, evaluation and commercialization of a probiotic strain. This study aims to develop identification methods for strains Bifidobacterium animalis subsp. lactis DSM 15954 and Bi-07 (Bi-07™) based on real-time PCR, targeting single nucleotide polymorphisms (SNPs). The SNPs were targeted by PCR assays with locked nucleic acid (LNA) probes, which is a novel application in probiotic identification. The assays were then validated following the guidelines for validating qualitative real-time PCR assays. Each assay was evaluated for specificity against 22 non-target strains including closely related Bifidobacterium animalis subsp. lactis strains and were found to achieve 100% true positive and 0% false positive rates. To determine reaction sensitivity and efficiency, three standard curves were established for each strain. Reaction efficiency values were 86, 91, and 90% (R square values &gt; 0.99), and 87, 84, and 86% (R square values &gt; 0.98) for B. animalis subsp. lactis DSM 15954 and Bi-07 assays, respectively. The limit of detection (LOD) was 5.0 picograms and 0.5 picograms of DNA for DSM 15954 and Bi-07 assays, respectively. Each assay was evaluated for accuracy using five samples tested at three different DNA concentrations and both assays proved to be highly repeatable and reproducible. Standard deviation of Cq values between two replicates was always below 1.38 and below 1.68 for DSM 15954 and Bi-07 assays, respectively. The assays proved to be applicable to mono-strain and multi-strain samples as well as for samples in various matrices of foods or dietary supplement ingredients. Overall, the methods demonstrated high specificity, sensitivity, efficiency and precision and broad applicability to sample, matrix and machine types. These methods facilitate strain level identification of the highly monophyletic strains B. animalis subsp. lactis DSM 15954 and Bi-07 to ensure probiotic efficacy and provide a strategy to identify other closely related probiotics organisms.

scholarly journals A real-time PCR method for detection of CpTI (Cowpea Trypsin Inhibitor) gene in the genetically modified rice originating from China

2015 ◽  
Vol 18 (2) ◽  
pp. 74-86
Author(s):  
Linh Thi My Nguyen ◽  
Thanh Nguyen Chu ◽  
Le Van Bui
Keyword(s):  
Real Time ◽  
Transgenic Rice ◽  
Trypsin Inhibitor ◽  
Real Time Pcr ◽  
Genetically Modified Organisms ◽  
Limit Of Detection ◽  
Genetically Modified ◽  
Positive Control ◽  
Amplification Efficiency ◽  
Cowpea Trypsin Inhibitor

Labelling and traceability of genetically modified organisms (GMOs) are necessary for trade and regulation in the world and Vietnam. Cowpea Trypsin Inhibitor (CpTI) gene encodes a trypsin inhibitor which is considered as a suitable candidate for developing insect-resistant transgenic plants, especially transgenic rice lines originating from China. In this study, we established a real-time PCR protocol to detect the CpTI gene in transgenic rice. The protocol with CpTI-F and CpTI-R primers, 300 nM primers, 0.5 X SYBR Green I, annealing temperature at 62 0C showed the best results. Amplification efficiency is 94.64 % and the limit of detection is 50 copies. Moreover, PCR product of CpTI gene was cloned into pBluescript plasmid using as a positive control

Salmonella detection in poultry samples

2012 ◽  
Vol 40 (06) ◽  
pp. 383-389 ◽  
Author(s):  
D. Sommer ◽  
D. Enderlein ◽  
A. Antakli ◽  
H. Schönenbrücher ◽  
J. Slaghuis ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Detection System ◽  
Cell Damage ◽  
Culture Method ◽  
Reference Laboratory ◽  
Salmonella Spp ◽  
Culture Methods ◽  
Fecal Samples

Summary Study: The efficiency of two commercial PCR methods based on real-time technology, the foodproof® Salmonella detection system and the BAX® PCR Assay Salmonella system was compared to standardized culture methods (EN ISO 6579:2002 – Annex D) for the detection of Salmonella spp. in poultry samples. Material and methods: Four sample matrices (feed, dust, boot swabs, feces) obtained directly from poultry flocks, as well as artificially spiked samples of the same matrices, were used. All samples were tested for Salmonella spp. using culture methods first as the gold standard. In addition samples spiked with Salmonella Enteridis were tested to evaluate the sensitivity of both PCR methods. Furthermore all methods were evaluated in an annual ring-trial of the National Salmonella Reference Laboratory of Germany. Results: Salmonella detection in the matrices feed, dust and boot swabs were comparable in both PCR systems whereas the results from feces differed markedly. The quality, especially the freshness, of the fecal samples had an influence on the sensitivity of the real-time PCR and the results of the culture methods. In fresh fecal samples an initial spiking level of 100 cfu/25 g Salmonella Enteritidis was detected. Two-days-dried fecal samples allowed the detection of 14 cfu/25 g. Both real-time PCR protocols appear to be suitable for the detection of Salmonella spp. in all four matrices. The foodproof® system detected eight samples more to be positive compared to the BAX® system, but had a potential false positive result in one case. In 7-days-dried samples none of the methods was able to detect Salmonella likely through letal cell damage. Clinical relevance: In general the advantage of PCR analyses over the culture method is the reduction of working time from 4–5 days to only 2 days. However, especially for the analysis of fecal samples official validation should be conducted according to the requirement of EN ISO 6579:2002 – Annex D.

scholarly journals Detection of Mycobacterium avium Subspecies Paratuberculosis in Pooled Fecal Samples by Fecal Culture and Real-Time PCR in Relation to Bacterial Density

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1605
Author(s):  
Annika Wichert ◽  
Esra Einax ◽  
Natalie Hahn ◽  
Anne Klassen ◽  
Karsten Donat
Keyword(s):  
Real Time ◽  
Mycobacterium Avium ◽  
Detection Probability ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Positive Association ◽  
Significant Loss ◽  
Positive Sample ◽  
Bacterial Density ◽  
Fecal Samples ◽  
Real Time Quantitative Pcr

Within paratuberculosis control programs Mycobacterium avium subsp. paratuberculosis (MAP)-infected herds have to be detected with minimum effort but with sufficient reliability. We aimed to evaluate a combination of random sampling (RS) and pooling for the detection of MAP-infected herds, simulating repeated RS in imitated dairy herds (within-herd prevalence 1.0%, 2.0%, 4.3%). Each RS consisted of taking 80 out of 300 pretested fecal samples, and five or ten samples were repeatedly and randomly pooled. All pools containing at least one MAP-positive sample were analyzed by culture and real-time quantitative PCR (qPCR). The pool detection probability was 47.0% or 45.9% for pools of size 5 or 10 applying qPCR and slightly lower using culture. Combining these methods increased the pool detection probability. A positive association between bacterial density in pools and pool detection probability was identified by logistic regression. The herd-level detection probability ranged from 67.3% to 84.8% for pools of size 10 analyzed by both qPCR and culture. Pools of size 10 can be used without significant loss of sensitivity compared with pools of size 5. Analyzing randomly sampled and pooled fecal samples allows the detection of MAP-infected herds, but is not recommended for one-time testing in low prevalence herds.

scholarly journals Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis

PLoS ONE ◽  
2015 ◽  
Vol 10 (11) ◽  
pp. e0143444 ◽  
Author(s):  
Yong Zhao ◽  
Guilian Li ◽  
Chongyun Sun ◽  
Chao Li ◽  
Xiaochen Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Locked Nucleic Acid ◽  
Pcr Assay ◽  
Locked Nucleic Acid Probe
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