scholarly journals Development of a high-throughput real-time PCR system for detection of enzootic pathogens in pigs

2019 ◽  
Vol 32 (1) ◽  
pp. 51-64 ◽  
Author(s):  
Nicole B. Goecke ◽  
Charlotte K. Hjulsager ◽  
Jesper S. Krog ◽  
Kerstin Skovgaard ◽  
Lars E. Larsen
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Pathogen Detection ◽  
Detection System ◽  
Working Hours ◽  
Negative Influence ◽  
Optimal Choice ◽  
National Veterinary Institute ◽  
Wide Range

Respiratory and intestinal diseases in pigs can have significant negative influence on productivity and animal welfare. A wide range of real-time PCR (rtPCR) assays are used in our laboratory (National Veterinary Institute, Technical University of Denmark) for pathogen detection, and PCR analyses are performed on traditional rtPCR platforms in which a limited number of samples can be analyzed per day given limitations in equipment and personnel. To mitigate these restrictions, rtPCR assays have been optimized for the high-throughput rtPCR BioMark platform (Fluidigm). Using this platform, we developed a high-throughput detection system that can be used for simultaneous examination of 48 samples with detection specificity for 18 selected respiratory and enteric viral and bacterial pathogens of high importance to Danish pig production. The rtPCR assays were validated and optimized to run under the same reaction conditions using a BioMark 48.48 dynamic array (DA) integrated fluidic circuit chip, and the sensitivity and specificity were assessed by testing known positive samples. Performance of the 48.48DA was similar to traditional rtPCR analysis, and the specificity of the 48.48DA was high. Application of the high-throughput platform has resulted in a significant reduction in cost and working hours and has provided production herds with a new innovative service with the potential to facilitate the optimal choice of disease control strategies such as vaccination and treatment.

scholarly journals High-throughput HTLV-1 proviral DNA detection system using a nucleic acid extraction robot and real-time PCR detection.

2001 ◽  
Vol 47 (3) ◽  
pp. 378-383 ◽  
Author(s):  
Chieko Matsumoto ◽  
Rieko Shiozawa ◽  
Shigeki Mitsunaga ◽  
Akiko Ichikawa ◽  
Rika Ishiwatari ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Detection System ◽  
Dna Detection ◽  
Pcr Detection ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Proviral Dna

scholarly journals Bovine ticks harbour a diverse array of microorganisms in Pakistan

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Abdul Ghafar ◽  
Alejandro Cabezas-Cruz ◽  
Clemence Galon ◽  
Dasiel Obregon ◽  
Robin B. Gasser ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Distribution Patterns ◽  
Diagnostic Methods ◽  
Mixed Infections ◽  
Ecological Zones ◽  
Wide Range ◽  
Relationship Of

Abstract Background Ticks and tick-borne pathogens (TTBP) are a major constraint to livestock production in Pakistan; despite a high prevalence of TTBPs, knowledge on the capacity of Pakistani ticks to carry pathogens and endosymbionts is limited. Furthermore, mixed infections with multiple microorganisms further complicate and limit the detection potential of traditional diagnostic methods. The present study investigated the tick-borne microorganisms in bovine ticks in Pakistan, employing a high-throughput microfluidic real-time PCR based technique. Methods Ticks were collected from clinically healthy cattle (n = 116) and water buffaloes (n = 88) from 30 villages across six districts located in five agro-ecological zones (AEZs) of Pakistan from September to November 2017. The microfluidic real-time PCR was used to test the genomic DNA of individual ticks for the presence of 27 bacterial and eight parasitic microorganisms. Phylogenetic methods were used to assess the genetic relationship of DNA sequences determined herein. Results PCR detected DNA of at least one microorganism in each of 221 ticks tested (94.4%, 221/234). DNA-based detection inferred that single pathogens/endosymbionts were the most common (43.4%, 96/221) followed by double (38.9%, 86/221), triple (14.5%, 32/221), quadruple (2.3%, 5/221) and quintuple (0.9%, 2/221) mixed infections. Piroplasms (Babesia/Theileria spp.) were the most prevalent (31.6%, 74/234), followed by Ehrlichia spp. (20%, 47/234) and Anaplasma marginale (7.7%, 18/234). Anaplasma phagocytophilum, A. ovis, A. centrale, Babesia ovis, Borrelia spp., Rickettsia spp., R. massiliae, Bartonella spp. and Hepatozoon spp. were also detected. Endosymbionts such as Francisella-like (91.5%, 214/234) and Coxiella-like (1.3%, 3/234) organisms were also detected in ticks. The highest diversity of microorganisms was detected in Hyalomma anatolicum ticks (test-positive for 14/14 microorganisms), followed by Rhipicephalus microplus (4/14), Hy. hussaini (3/14) and Rh. annulatus (2/14). Ticks collected from cattle carried significantly more frequently piroplasms (41.2%, 54/131; P < 0.05) than those from buffaloes (19.4%, 20/103). However, the overall prevalence of microorganisms did not vary significantly among ticks from the two host species as well as across different AEZs. Conclusions To our knowledge, this is the first study to investigate a wide range of tick-borne microorganisms in bovine ticks using a high-throughput diagnostic method from different AEZs in Pakistan. These findings will aid in establishing the distribution patterns and the control of tick-borne pathogens of bovines in Pakistan.

scholarly journals Rapid high throughput SYBR green assay for identifying the malaria vectors Anopheles arabiensis, Anopheles coluzzii and Anopheles gambiae s.s. Giles

2018 ◽  
Author(s):  
Joseph Chabi ◽  
Arjen Van’t Hof ◽  
Louis K. N’dri ◽  
Alex Datsomor ◽  
Dora Okyere ◽  
...  
Keyword(s):  
Anopheles Gambiae ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Sybr Green ◽  
Molecular Diagnostic ◽  
Anopheles Coluzzii ◽  
Wide Range ◽  
Pcr Method ◽  
Dissociation Curves

AbstractThe Anopheles gambiae sensu lato species complex consists of a number of cryptic species with different habitats and behaviours. These morphologically indistinct species are identified by chromosome banding and molecular diagnostic techniques which are still under improvement even though the current SINE method for identification between An. coluzzii and An. gambiae works reliably. This study describes a refinement of the SINE method to increase sensitivity and high throughput method for the identification of both species and An. arabiensis using amplicon dissociation characteristics.Field collected samples, laboratory reared colonies and crossed specimens of the two species were used for the design of the protocol. An. gambiae, An. coluzzii, and hybrids of the two species were provided by the insectary of Vestergaard-NMIMR Vector Labs at the Noguchi Memorial Institute for Medical Research (Ghana) and An. arabiensis from Kenya. Samples were first characterised using conventional SINE PCR method, and further assayed using SYBR green, an intercalating fluorescent dye.The three species and hybrids were clearly differentiated using the melting temperature of the dissociation curves, with derivative peaks at 72 Celsius for An. arabiensis, 75°C for An. gambiae and 86°C for An. coluzzii. The hybrids (An. gambiae / An. coluzzii) showed both peaks. This work is the first to describe a SYBR green real time PCR method for the characterization of An. arabiensis, An. gambiae and An. coluzzii and was purposely designed for basic melt-curve analysis (rather than high-resolution melt-curve) to allow it to be used on a wide range of real-time PCR machines.

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.

Rapid detection and quantitation of hepatitis B virus DNA by real-time PCR using a new fluorescent (FRET) detection system

2004 ◽  
Vol 30 (2) ◽  
pp. 191-195 ◽  
Author(s):  
Sani Hussein Aliyu ◽  
Muktar Hassan Aliyu ◽  
Hamisu M Salihu ◽  
Surendra Parmar ◽  
Hamid Jalal ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Detection System ◽  
Hepatitis B Virus Dna ◽  
B Virus

scholarly journals Diversity of KIR genes and their HLA-C ligands in Ugandan populations with historically varied malaria transmission intensity

2021 ◽  
Author(s):  
Stephen Tukwasibwe ◽  
James A. Traherne ◽  
Olympe Chazara ◽  
Jyothi Jayaraman ◽  
John Trowsdale ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Malaria Transmission ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Association Studies ◽  
Transmission Intensity ◽  
Malaria Transmission Intensity ◽  
Significant Difference ◽  
Pcr Method

Abstract Background: Malaria is one of the most serious infectious diseases in the world. The malaria burden is greatly affected by human immunity, and immune responses vary between populations. Genetic diversity in KIR and HLA-C genes, which are important in immunity to infectious diseases, is likely to play a role in this heterogeneity. Several studies have shown that KIR and HLA-C genes influence the immune response to viral infections, but few studies have examined the role of KIR and HLA-C in malaria infection, and these have used low-resolution genotyping. The aim of this study was to determine whether genetic variation in KIR and their HLA-C ligands differ in Ugandan populations with historically varied malaria transmission intensity using more comprehensive genotyping approaches.Methods: High throughput multiplex quantitative real-time PCR method was used to genotype KIR genetic variants and copy number variation and a high-throughput real-time PCR method was developed to genotype HLA-C1 and C2 allotypes for 1,344 participants, aged 6 months to 10 years, enrolled from Ugandan populations with historically high (Tororo District), medium (Jinja District) and low (Kanungu District) malaria transmission intensity. Results: The prevalence of KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes was significantly lower in populations from Kanungu compared to Tororo (7.6% vs. 13.2%: p=0.006, 57.2% vs. 66.4%: p=0.005, 33.2% vs. 46.6%: p<0.001 and 19.7% vs. 26.7%: p=0.014 respectively) or Jinja (7.6% vs.18.1%: p<0.001, 57.2% vs. 63.8%: p=0.048, 33.2% vs. 43.5%: p=0.002 and 19.7% vs. 30.4%: p<0.001 respectively). The prevalence of homozygous HLA-C2 was significantly higher in populations from Kanungu (31.6%) compared to Jinja (21.4%), p=0.043, with no significant difference between Kanungu and Tororo (26.7%), p=0.296. Conclusions: The KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes may partly explain differences in transmission intensity of malaria since these genes have been positively selected for in places with historically high malaria transmission intensity. The high-throughput multiplex real-time HLA-C genotyping PCR method developed will be useful in disease association studies involving large cohorts.

scholarly journals Estimating Clinically Relevant Cut-Off Values for a High-Throughput Quantitative Real-Time PCR Detecting Bacterial Respiratory Pathogens in Cattle

2021 ◽  
Vol 8 ◽  
Author(s):  
Alicia F. Klompmaker ◽  
Maria Brydensholt ◽  
Anne Marie Michelsen ◽  
Matthew J. Denwood ◽  
Carsten T. Kirkeby ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Pasteurella Multocida ◽  
Age Groups ◽  
Therapeutic Interventions ◽  
Respiratory Pathogens ◽  
Logistic Regression Models ◽  
Histophilus Somni ◽  
Nasal Swabs

Bovine respiratory disease (BRD) results from interactions between pathogens, environmental stressors, and host factors. Obtaining a diagnosis of the causal pathogens is challenging but the use of high-throughput real-time PCR (rtPCR) may help target preventive and therapeutic interventions. The aim of this study was to improve the interpretation of rtPCR results by analysing their associations with clinical observations. The objective was to develop and illustrate a field-data driven statistical method to guide the selection of relevant quantification cycle cut-off values for pathogens associated with BRD for the high-throughput rtPCR system “Fluidigm BioMark HD” based on nasal swabs from calves. We used data from 36 herds enrolled in a Danish field study where 340 calves within pre-determined age-groups were subject to clinical examination and nasal swabs up to four times. The samples were analysed with the rtPCR system. Each of the 1,025 observation units were classified as sick with BRD or healthy, based on clinical scores. The optimal rtPCR results to predict BRD were investigated for Pasteurella multocida, Mycoplasma bovis, Histophilus somni, Mannheimia haemolytica, and Trueperella pyogenes by interpreting scatterplots and results of mixed effects logistic regression models. The clinically relevant rtPCR cut-off suggested for P. multocida and M. bovis was ≤ 21.3. For H. somni it was ≤ 17.4, while no cut-off could be determined for M. haemolytica and T. pyogenes. The demonstrated approach can provide objective support in the choice of clinically relevant cut-offs. However, for robust performance of the regression model sufficient amounts of suitable data are required.

scholarly journals Massively parallel single-molecule telomere length measurement with digital real-time PCR

2020 ◽  
Vol 6 (34) ◽  
pp. eabb7944 ◽  
Author(s):  
Yongqiang Luo ◽  
Ramya Viswanathan ◽  
Manoor Prakash Hande ◽  
Amos Hong Pheng Loh ◽  
Lih Feng Cheow
Keyword(s):  
Telomere Length ◽  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Real Time Pcr ◽  
Length Distribution ◽  
Telomere Maintenance ◽  
Massively Parallel ◽  
Primary Tumors ◽  
Absolute Length

Telomere length is a promising biomarker for age-associated diseases and cancer, but there are still substantial challenges to routine telomere analysis in clinics because of the lack of a simple and rapid yet scalable method for measurement. We developed the single telomere absolute-length rapid (STAR) assay, a novel high-throughput digital real-time PCR approach for rapidly measuring the absolute lengths and quantities of individual telomere molecules. We show that this technique provides the accuracy and sensitivity to uncover associations between telomere length distribution and telomere maintenance mechanisms in cancer cell lines and primary tumors. The results indicate that the STAR assay is a powerful tool to enable the use of telomere length distribution as a biomarker in disease and population-wide studies.

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