scholarly journals Sensitive detection of porcine circovirus 3 by droplet digital PCR

2019 ◽  
Vol 31 (4) ◽  
pp. 604-607
Author(s):  
Yuqi Liu ◽  
Hecheng Meng ◽  
Lei Shi ◽  
Lili Li
Keyword(s):  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Porcine Circovirus ◽  
Analytical Sensitivity ◽  
Swine Industry ◽  
Potential Threat ◽  
Emerging Virus ◽  
Clinical Detection ◽  
Porcine Circovirus 3 ◽  
High Degree

Porcine circovirus 3 (PCV-3) is a newly emerging virus that poses a potential threat to the swine industry. We developed a sensitive assay utilizing droplet digital PCR (ddPCR) to detect PCV-3. Specificity of the assay was confirmed by the failure of amplification of DNA of other relevant viruses. The detection limit for ddPCR was 1 copy/μL, 10 times greater sensitivity than TaqMan real-time PCR (rtPCR). Both methods showed a high degree of linearity, although TaqMan rtPCR showed less sensitivity than ddPCR for clinical detection. Our findings indicate that ddPCR might offer faster and improved analytical sensitivity for PCV-3 detection.

Development of a droplet digital PCR assay for sensitive detection of porcine circovirus 3

2019 ◽  
Vol 43 ◽  
pp. 50-57 ◽  
Author(s):  
Yongning Zhang ◽  
Zhou Zhang ◽  
Zhanying Wang ◽  
Zili Wang ◽  
Caixia Wang ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Sensitive Detection ◽  
Porcine Circovirus ◽  
Pcr Assay ◽  
Porcine Circovirus 3

scholarly journals Evolution and Genetic Diversity of Porcine Circovirus 3 in China

Viruses ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 786 ◽  
Author(s):  
Ye Chen ◽  
Quanming Xu ◽  
Hong Chen ◽  
Xian Luo ◽  
Qi Wu ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Eastern China ◽  
Porcine Circovirus ◽  
Recent Common Ancestor ◽  
Fujian Province ◽  
Tissue Samples ◽  
Genetic Characteristics ◽  
Most Recent Common Ancestor ◽  
Porcine Circovirus 3 ◽  
High Degree

The identification of a new circovirus (Porcine Circovirus 3, PCV3) has raised concern because its impact on swine health is not fully known. In Fujian Province in eastern China, even its circulating status and genetic characteristics are unclear. Here, we tested 127 tissue samples from swine from Fujian Province that presented respiratory symptoms. All of the PCV3 positive samples were negative for many other pathogens involved in respiratory diseases like PCV2, PRRSV, and CSFV, suggesting that PCV3 is potentially pathogenic. From phylogenetic analysis, PCV3 strains are divided into two main clades and five sub-clades; PCV3a-1, PCV3a-2, PCV3a-3, PCV3b-1, and PCV3b-2. Our identified strains belong to genotypes PCV3a-1, PCV3a-2, PCV3a-3, and PCV3b-2, indicating a high degree of genetic diversity of PCV3 in Fujian province until 2019. Interestingly, we found the time of the most recent common ancestor (tMRCA) of PCV3 was dated to the 1950s, and PCV3 has a similar evolutionary rate as PCV2 (the main epidemic genotypes PCV2b and PCV2d). In addition, positive selection sites N56D/S and S77T/N on the capsid gene are located on the PCV3 antigen epitope, indicating that PCV3 is gradually adaptive in swine. In summary, our results provide important insights into the epidemiology of PCV3.

scholarly journals Acoustic array biochip combined with allele-specific PCR for multiple cancer mutation analysis in tissue and liquid biopsy

2021 ◽  
Author(s):  
Nikoletta Naoumi ◽  
Kleita Michaelidou ◽  
George Papadakis ◽  
Agapi E. Simaiaki ◽  
Roman Fernandez ◽  
...  
Keyword(s):  
Cost Effectiveness ◽  
Liquid Biopsy ◽  
Point Mutations ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Braf V600e ◽  
Analytical Sensitivity ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

Regular screening of cancerous point mutations is of importance to cancer management and treatment selection. Although excellent techniques like next-generation sequencing and droplet digital PCR are available, these are still lacking in speed, simplicity and cost-effectiveness. Here a new approach is presented where allele-specific PCR (AS-PCR) is combined with a novel High Fundamental Frequency Quartz Crystal Microbalance (HFF-QCM) array biosensor for the amplification and detection, respectively, of cancer point mutations. For the proof-of-concept, the method was applied to the screening of the BRAF V600E and KRAS G12D mutations in spiked-in and clinical samples. Regarding the BRAF target, an analytical sensitivity of 0.01%, i.e., detection of 1 mutant copy of genomic DNA in an excess of 104 wild type molecules, was demonstrated; moreover, quantitative results during KRAS detection were obtained when an optimized assay was employed with a sensitivity of 0.05%. The assays were validated using tissue and plasma samples obtained from melanoma, colorectal and lung cancer patients. Results are in full agreement with Sanger sequencing and droplet digital PCR, demonstrating efficient detection of BRAF and KRAS mutations in samples having an allele frequency below 1%. The high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness and compatibility with routine work-flow, hold promise for the implementation of this AS-PCR/acoustic methodology in clinical oncology as a tool for tissue and liquid biopsy.

scholarly journals Development and validation of direct PCR and quantitative PCR assays for the rapid, sensitive, and economical detection of porcine circovirus 3

2018 ◽  
Vol 30 (4) ◽  
pp. 538-544 ◽  
Author(s):  
Giovanni Franzo ◽  
Matteo Legnardi ◽  
Cinzia Centelleghe ◽  
Claudia M. Tucciarone ◽  
Mattia Cecchinato ◽  
...  
Keyword(s):  
Test Performance ◽  
Low Cost ◽  
High Sensitivity ◽  
Porcine Circovirus ◽  
Direct Pcr ◽  
Swine Industry ◽  
Perfect Agreement ◽  
Field Samples ◽  
Porcine Circovirus 3 ◽  
Pcr Targeting

Since the identification of species Porcine circovirus 2, the relevance of genus Circovirus has increased given its impact on the swine industry. A new species ( Porcine circovirus 3, PCV-3) has been detected in association with various clinical conditions. Consequently, there is an urgent need for reliable and widely accessible tests for both routine diagnostic and research purposes. We developed a direct PCR (requiring no DNA extraction) and a quantitative (q)PCR targeting the conserved rep gene to detect the PCV-3 genome. Test performance was assessed by testing 120 field samples within different matrices. Both methods were sensitive (detection of 10 viral genome/µL), specific, and repeatable. The substantially perfect agreement between the 2 assays strongly supports their high sensitivity and specificity. The low cost and short processing time of the direct PCR protocol, together with the reliable quantitative results provided by qPCR, support the establishment of common testing guidelines.

scholarly journals Current Knowledge on Porcine circovirus 3 (PCV-3): A Novel Virus With a Yet Unknown Impact on the Swine Industry

2018 ◽  
Vol 5 ◽  
Author(s):  
Francini Klaumann ◽  
Florencia Correa-Fiz ◽  
Giovanni Franzo ◽  
Marina Sibila ◽  
José I. Núñez ◽  
...  
Keyword(s):  
Current Knowledge ◽  
Porcine Circovirus ◽  
Swine Industry ◽  
Novel Virus ◽  
Porcine Circovirus 3

scholarly journals Direct Comparison of SARS-CoV-2 Analytical Limits of Detection across Seven Molecular Assays

2020 ◽  
Vol 58 (9) ◽  
Author(s):  
Becky Fung ◽  
Allan Gopez ◽  
Venice Servellita ◽  
Shaun Arevalo ◽  
Coral Ho ◽  
...  
Keyword(s):  
High Throughput ◽  
Extraction Method ◽  
Point Of Care ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Analytical Sensitivity ◽  
Limits Of Detection ◽  
Molecular Assays ◽  
Performance Metric ◽  
Roche Cobas

ABSTRACT Analytical sensitivity for SARS-CoV-2 detection is a key performance metric for the evaluation of viral detection assays. We determined analytical limits of detection for seven SARS-CoV-2 assays using serial dilutions of pooled patient material quantified with droplet digital PCR. Limits of detection ranged from ≤10 to 74 copies/ml for commercial high-throughput laboratory analyzers (Roche Cobas, Abbott m2000, and Hologic Panther Fusion) and 167 to 511 copies/ml for sample-to-answer (DiaSorin Simplexa, GenMark ePlex) and point-of-care instruments (Abbott ID NOW). The CDC assay yielded limits of detection ranging from 85 to 499 copies/ml, depending on the extraction method and thermocycler used. These results can help to inform the assay choice for testing approaches to manage the current COVID-19 outbreak.

scholarly journals Development of a droplet digital PCR for detection and quantification of porcine epidemic diarrhea virus

2020 ◽  
Vol 32 (4) ◽  
pp. 572-576 ◽  
Author(s):  
Wei W. Cao ◽  
Dong S. He ◽  
Zhen J. Chen ◽  
Yu Z. Zuo ◽  
Xun Chen ◽  
...  
Keyword(s):  
Porcine Epidemic Diarrhea Virus ◽  
Fold Increase ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Economic Losses ◽  
Swine Industry ◽  
Diarrhea Virus ◽  
Viral Loads ◽  
Promising Tool ◽  
Porcine Epidemic Diarrhea

Porcine epidemic diarrhea, a disease caused by porcine epidemic diarrhea virus (PEDV), results in large economic losses to the global swine industry. To manage this disease effectively, it is essential to detect PEDV early and accurately. We developed a sensitive and accurate droplet digital PCR (ddPCR) assay to detect PEDV. The optimal primer-to-probe concentration and melting temperature were identified as 300:200 nM and 59.2°C, respectively. The specificity of the ddPCR assay was confirmed by negative test results for common swine pathogens. The detection limit for the ddPCR was 0.26 copies/μL, which is a 5.7-fold increase in sensitivity compared to that of real-time PCR (rtPCR). Both ddPCR and rtPCR assays exhibited good linearity, although ddPCR provided higher sensitivity for clinical detection compared to that of rtPCR. Our ddPCR methodology provides a promising tool for evaluating the PEDV viral load when used for clinical testing, particularly for detecting samples with low-copy viral loads.

scholarly journals Genetic Diversity and Prevalence of Porcine Circovirus Type 2 in China During 2000-2019

2021 ◽  
Vol 8 ◽  
Author(s):  
Ning Li ◽  
Jing Liu ◽  
Jiali Qi ◽  
Feng Hao ◽  
Lei Xu ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Wide Distribution ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Amino Acid Sequences ◽  
Porcine Circovirus ◽  
Economic Losses ◽  
Swine Industry ◽  
Emerging Virus

As the major pathogen for porcine circovirus-associated disease (PCVAD), porcine circovirus type 2 (PCV2) is no longer treated as an emerging virus anymore. The wide distribution of PCV2 infection in China causes huge economic losses in the swine industry. Currently, it is generally believed that PCV2 has eight genotypes (PCV2a to PCV2h), with PCV2a, PCV2b, and PCV2d being widely distributed. To comprehensively explore the genetic diversity and prevalence of PCV2 in China, PCV-2 sequences submitted from China in the GenBank database were retrieved. With a total of 714 PCV2 strains were retrieved, we found that early-submitted PCV2 sequences were mainly collected from coastal provinces in the southeast part of China, which may indicate PCV2 was initially circulating in those regions. From 2002 to 2008, PCV2b was the dominant prevalent genotype in those retrieved sequences. From 2009, PCV2d became the dominant genotype in those sequences, dropping a hint that a potential shift of PCV2b to PCV2d might occur in 2009, which is similar to the patterns at the global level. In addition to the PCV2a, PCV2b, and PCV2d genotypes, novel strains were also characterized. We further revealed that the amino acid sequences consistency of PCV2a Cap is higher than those in other genotypes. Together, this study provided clues for the possible prevalent genotypes and dynamics of genetic diversity in China from 2000 to 2019.

scholarly journals Genetic Diversity of Porcine Circovirus 3 Strains and the First Detection of Two Different PCV3 Strains Coinfecting the Same Host in Minas Gerais, Brazil

2021 ◽  
Author(s):  
Viviane Sisdelli Assao ◽  
Marcus Rebouças Santos ◽  
Nívia Carolina Lopes Rosado ◽  
Gustavo Costa Bressan ◽  
Juliana Lopes Rangel Fietto ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Real Time Pcr ◽  
Growing Pigs ◽  
Porcine Circovirus ◽  
Quantitative Real Time Pcr ◽  
Swine Industry ◽  
Pig Farms ◽  
Weaning Pigs ◽  
Porcine Circovirus 3 ◽  
First Time

Abstract Porcine circovirus 3 (PCV3) is a recently emerged circovirus discovered in 2016, which since then has drawn the attention of the swine industry worldwide. In this study, we evaluated the genetic diversity of PCV3 strains in pig farms. A total of 261 samples from sows, weaning pigs, growing pigs, and stillborn/mummified fetuses were analyzed by quantitative real-time PCR. The results revealed that PCV3 strains have at least two main lineages circulating in Brazil. For the first time, it was possible to detect the presence of two different PCV3 strains in the same host.

scholarly journals Comparison of droplet digital PCR and quantitative PCR for the detection of Salmonella and its application for river sediments

2017 ◽  
Vol 15 (4) ◽  
pp. 505-508 ◽  
Author(s):  
Gulshan Singh ◽  
Ayanda Sithebe ◽  
Abimbola M. Enitan ◽  
Sheena Kumari ◽  
Faizal Bux ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Informal Settlement ◽  
Quantitative Polymerase Chain Reaction ◽  
River Sediments ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Analytical Sensitivity ◽  
Aquatic Sediments ◽  
Pcr Inhibitors ◽  
Copy Numbers

Despite advances in microbial detection that quantitative polymerase chain reaction (qPCR) has led to, complex environmental samples, such as sediments, remain a challenge due to presence of PCR inhibitors. Aquatic sediments accumulate particle-bound microbial contaminants and thereby reflect a cumulative microbial load over time. The relatively new droplet digital PCR (ddPCR) has emerged as a direct quantitative method, highly tolerant to PCR inhibitors and relinquishing the necessity for calibration/standard curves. Information is virtually absent where ddPCR has been applied to detect pathogenic organisms in aquatic sediments. This study compared the efficacy of ddPCR with qPCR, for quantification of Salmonella in sediments from the Palmiet River near an informal settlement in Durban, South Africa. ddPCR significantly improved both analytical sensitivity and detection of low concentrations of Salmonella as compared to qPCR. The expected copy numbers measured from both qPCR and ddPCR showed good R2 values (0.999 and 0.994, respectively). The site mostly affected by the informal settlements exhibited Salmonella in the range of 255 ± 37 and 818 ± 30 Salmonella/g (p ≤ 0.0001) in qPCR and ddPCR, respectively. The improved detection of Salmonella in sediments with ddPCR makes it a promising technical method for the quantification of Salmonella in multifarious environmental samples.

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