Detectability vs. time and costs in pooled DNA extraction of cutaneous swabs: a study on the amphibian chytrid fungi

2019 ◽  
Vol 40 (1) ◽  
pp. 29-39 ◽  
Author(s):  
Joana Sabino-Pinto ◽  
E. Tobias Krause ◽  
Molly C. Bletz ◽  
An Martel ◽  
Frank Pasmans ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Pathogen Detection ◽  
Batrachochytrium Dendrobatidis ◽  
Quantitative Polymerase Chain Reaction ◽  
Large Fraction ◽  
Work Load ◽  
Detection Sensitivity ◽  
Pooled Dna ◽  
Reaction Volumes ◽  
User Friendly

Abstract Epidemiology relies on understanding the distribution of pathogens which often can be detected through DNA-based techniques, such as quantitative Polymerase Chain Reaction (qPCR). Typically, the DNA of each individual sample is separately extracted and undergoes qPCR analysis. However, when performing field surveys and long-term monitoring, a large fraction of the samples is generally expected to be negative, especially in geographical areas still considered free of the pathogen. If pathogen detection within a population – rather than determining its individual prevalence – is the focus, work load and monetary costs can be reduced by pooling samples for DNA extraction. We test and refine a user-friendly technique where skin swabs can be pooled during DNA extraction to detect the amphibian chytrid fungi, Batrachochytrium dendrobatidis and B. salamandrivorans (Bsal). We extracted pools with different numbers of samples (from one to four swabs), without increasing reaction volumes, and each pool had one sample inoculated with a predetermined zoospore amount. Pool size did not reduce the ability to detect the two fungi, except if inoculated with extremely low zoospore amounts (one zoospore). We confirm that pooled DNA extraction of cutaneous swabs can substantially reduce processing time and costs without minimizing detection sensitivity. This is of relevance especially for the new emerging pathogen Bsal, for which pooled DNA extraction had so far not been tested and massive monitoring efforts in putatively unaffected regions are underway.

scholarly journals Validation of an eDNA-based method for the detection of wildlife pathogens in water

2020 ◽  
Vol 141 ◽  
pp. 171-184
Author(s):  
N Sieber ◽  
H Hartikainen ◽  
C Vorburger
Keyword(s):  
Water Samples ◽  
Pathogen Detection ◽  
Batrachochytrium Dendrobatidis ◽  
Tap Water ◽  
Pathogenic Fungus ◽  
Environmental Dna ◽  
Detection Sensitivity ◽  
Detection Rates ◽  
Filtration Step ◽  
Wildlife Pathogens

Monitoring the occurrence and density of parasites and pathogens can identify high infection-risk areas and facilitates disease control and eradication measures. Environmental DNA (eDNA) techniques are increasingly used for pathogen detection due to their relative ease of application. Since many factors affect the reliability and efficacy of eDNA-based detection, rigorous validation and assessment of method limitations is a crucial first step. We evaluated an eDNA detection method using in situ filtration of large-volume water samples, developed to detect and quantify aquatic wildlife parasites by quantitative PCR (qPCR). We assessed method reliability using Batrachochytrium dendrobatidis, a pathogenic fungus of amphibians and the myxozoan Tetracapsuloides bryosalmonae, causative agent of salmonid proliferative kidney disease, in a controlled experimental setup. Different amounts of parasite spores were added to tanks containing either clean tap water or water from a semi-natural mesocosm community. Overall detection rates were higher than 80%, but detection was not consistent among replicate samples. Within-tank variation in detection emphasises the need for increased site-level replication when dealing with parasites and pathogens. Estimated parasite DNA concentrations in water samples were highly variable, and a significant increase with higher spore concentrations was observed only for B. dendrobatidis. Despite evidence for PCR inhibition in DNA extractions from mesocosm water samples, the type of water did not affect detection rates significantly. Direct spiking controls revealed that the filtration step reduced detection sensitivity. Our study identifies sensitive quantification and sufficient replication as major remaining challenges for the eDNA-based methods for detection of parasites in water.

scholarly journals Wildlife pathogen detection: evaluation of alternative DNA extraction protocols

2021 ◽  
Author(s):  
Vasiliki Mantzana-Oikonomaki ◽  
Martine Maan ◽  
Joana Sabino-Pinto
Keyword(s):  
Dna Extraction ◽  
Pathogen Detection ◽  
Detection Efficiency ◽  
Quantitative Polymerase Chain Reaction ◽  
Wildlife Disease ◽  
False Negatives ◽  
Optimal Method ◽  
Spin Column ◽  
Pre Treatment ◽  
Wildlife Pathogens

Abstract Accurate detection of wildlife pathogens is critical in wildlife disease research. False negatives or positives can have catastrophic consequences for conservation and disease-mitigation decisions. Quantitative polymerase chain reaction is commonly used for molecular detection of wildlife pathogens. The reliability of this method depends on the effective extraction of the pathogen’s DNA from host samples. A wildlife disease that has been in the centre of conservationist’s attention is the amphibian disease Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Here, we compare the efficiency of a spin column extraction kit (QIAGEN), commonly used in Bd DNA extraction, to an alternative spin column kit (BIOKÈ) used in extractions from other types of samples, which is considerably cheaper but not typically used for Bd DNA extraction. Additionally, we explore the effect of an enzymatic pre-treatment on detection efficiency. Both methods showed similar efficiency when extracting Bd DNA from zoospores from laboratory-created cell-cultures, as well as higher efficiency when combined with the enzymatic pre-treatment. Our results indicate that selecting the optimal method for DNA extraction is essential to ensure minimal false negatives and reduce project costs.

Clin-mNGS: Automated Pipeline for Pathogen Detection from Clinical Metagenomic Data

2020 ◽  
Vol 15 ◽  
Author(s):  
Akshatha Prasanna ◽  
Vidya Niranjan
Keyword(s):  
Antimicrobial Resistance ◽  
High Performance ◽  
Pathogen Detection ◽  
Bacterial Species ◽  
Workflow Management ◽  
Metagenomic Data ◽  
Antimicrobial Resistance Genes ◽  
Culture Independent ◽  
Automated Pipeline ◽  
User Friendly

Background: Since bacteria are the earliest known organisms, there has been significant interest in their variety and biology, most certainly concerning human health. Recent advances in Metagenomics sequencing (mNGS), a culture-independent sequencing technology have facilitated an accelerated development in clinical microbiology and our understanding of pathogens. Objective: For the implementation of mNGS in routine clinical practice to become feasible, a practical and scalable strategy for the study of mNGS data is essential. This study presents a robust automated pipeline to analyze clinical metagenomic data for pathogen identification and classification. Method: The proposed Clin-mNGS pipeline is an integrated, open-source, scalable, reproducible, and user-friendly framework scripted using the Snakemake workflow management software. The implementation avoids the hassle of manual installation and configuration of the multiple command-line tools and dependencies. The approach directly screens pathogens from clinical raw reads and generates consolidated reports for each sample. Results: The pipeline is demonstrated using publicly available data and is tested on a desktop Linux system and a High-performance cluster. The study compares variability in results from different tools and versions. The versions of the tools are made user modifiable. The pipeline results in quality check, filtered reads, host subtraction, assembled contigs, assembly metrics, relative abundances of bacterial species, antimicrobial resistance genes, plasmid finding, and virulence factors identification. The results obtained from the pipeline are evaluated based on sensitivity and positive predictive value. Conclusion: Clin-mNGS is an automated Snakemake pipeline validated for the analysis of microbial clinical metagenomics reads to perform taxonomic classification and antimicrobial resistance prediction.

scholarly journals A Method For Parallel, Automated, Thermal Cycling of Submicroliter Samples

2001 ◽  
Vol 11 (3) ◽  
pp. 441-447
Author(s):  
Jonathan Nakane ◽  
David Broemeling ◽  
Roger Donaldson ◽  
Andre Marziali ◽  
Thomas D. Willis ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Thermal Cycling ◽  
High Throughput ◽  
Dna Analysis ◽  
Detection Efficiency ◽  
Large Fraction ◽  
Cycle Sequencing ◽  
High Throughput Dna Sequencing ◽  
Reaction Volumes ◽  
The Cost

A large fraction of the cost of DNA sequencing and other DNA-analysis processes results from the reagent costs incurred during cycle sequencing or PCR. In particular, the high cost of the enzymes and dyes used in these processes often results in thermal cycling costs exceeding $0.50 per sample. In the case of high-throughput DNA sequencing, this is a significant and unnecessary expense. Improved detection efficiency of new sequencing instrumentation allows the reaction volumes for cycle sequencing to be scaled down to one-tenth of presently used volumes, resulting in at least a 10-fold decrease in the cost of this process. However, commercially available thermal cyclers and automated reaction setup devices have inherent design limitations which make handling volumes of <1 μL extremely difficult. In this paper, we describe a method for thermal cycling aimed at reliable, automated cycling of submicroliter reaction volumes.

scholarly journals Variations in terrestrial arthropod DNA metabarcoding methods recovers robust beta diversity but variable richness and site indicators

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Teresita M. Porter ◽  
Dave M. Morris ◽  
Nathan Basiliko ◽  
Mehrdad Hajibabaei ◽  
Daniel Doucet ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Beta Diversity ◽  
Ecological Integrity ◽  
Sequence Variants ◽  
Key Indicator ◽  
Pooled Dna ◽  
Dna Metabarcoding ◽  
Arthropod Fauna ◽  
Reference Databases ◽  
Arthropod Biodiversity

AbstractTerrestrial arthropod fauna have been suggested as a key indicator of ecological integrity in forest systems. Because phenotypic identification is expert-limited, a shift towards DNA metabarcoding could improve scalability and democratize the use of forest floor arthropods for biomonitoring applications. The objective of this study was to establish the level of field sampling and DNA extraction replication needed for arthropod biodiversity assessments from soil. Processing 15 individually collected soil samples recovered significantly higher median richness (488–614 sequence variants) than pooling the same number of samples (165–191 sequence variants) prior to DNA extraction, and we found no significant richness differences when using 1 or 3 pooled DNA extractions. Beta diversity was robust to changes in methodological regimes. Though our ability to identify taxa to species rank was limited, we were able to use arthropod COI metabarcodes from forest soil to assess richness, distinguish among sites, and recover site indicators based on unnamed exact sequence variants. Our results highlight the need to continue DNA barcoding local taxa during COI metabarcoding studies to help build reference databases. All together, these sampling considerations support the use of soil arthropod COI metabarcoding as a scalable method for biomonitoring.

Evaluation of alternative DNA extraction processes and real-time PCR for detecting Cryptosporidium parvum in drinking water

2015 ◽  
Vol 15 (6) ◽  
pp. 1295-1303
Author(s):  
Gina H. Kimble ◽  
Vincent R. Hill ◽  
James E. Amburgey
Keyword(s):  
Drinking Water ◽  
Real Time ◽  
Dna Extraction ◽  
Cryptosporidium Parvum ◽  
Water Samples ◽  
Real Time Pcr ◽  
Extraction Procedure ◽  
The United States ◽  
Detection Sensitivity ◽  
Spin Column

USEPA Method 1623 is the standard method in the United States for the detection of Cryptosporidium in water samples, but quantitative real-time polymerase chain reaction (qPCR) is an alternative technique that has been successfully used to detect Cryptosporidium in aqueous matrices. This study examined various modifications to a commercial nucleic acid extraction procedure in order to enhance PCR detection sensitivity for Cryptosporidium. An alternative DNA extraction buffer allowed for qPCR detection at lower seed levels than a commercial extraction kit buffer. In addition, the use of a second spin column cycle produced significantly better detection (P = 0.031), and the volume of Tris–EDTA buffer significantly affected crossing threshold values (P = 0.001). The improved extraction procedure was evaluated using 10 L of tap water samples processed by ultrafiltration, centrifugation and immunomagnetic separation. Mean recovery for the sample processing method was determined to be 41% using microscopy and 49% by real-time PCR (P = 0.013). The results of this study demonstrate that real-time PCR can be an effective alternative for detecting and quantifying Cryptosporidium parvum in drinking water samples.

scholarly journals Development of an Efficient Real-Time Quantitative PCR Protocol for Detection ofXanthomonas arboricolapv. pruni inPrunusSpecies

2010 ◽  
Vol 77 (1) ◽  
pp. 89-97 ◽  
Author(s):  
Ana Palacio-Bielsa ◽  
Jaime Cubero ◽  
Miguel A. Cambra ◽  
Raquel Collados ◽  
Isabel M. Berruete ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Plant Material ◽  
Pathogen Detection ◽  
Plant Protection ◽  
The European Union ◽  
Real Time Quantitative Pcr ◽  
Field Samples ◽  
Mediterranean Plant ◽  
Different Origins

ABSTRACTXanthomonas arboricolapv. pruni, the causal agent of bacterial spot disease of stone fruit, is considered a quarantine organism by the European Union and the European and Mediterranean Plant Protection Organization (EPPO). The bacterium can undergo an epiphytic phase and/or be latent and can be transmitted by plant material, but currently, only visual inspections are used to certify plants as beingX. arboricolapv. pruni free. A novel and highly sensitive real-time TaqMan PCR detection protocol was designed based on a sequence of a gene for a putative protein related to an ABC transporter ATP-binding system inX. arboricolapv. pruni. Pathogen detection can be completed within a few hours with a sensitivity of 102CFU ml−1, thus surpassing the sensitivity of the existing conventional PCR. Specificity was assessed forX. arboricolapv. pruni strains from different origins as well as for closely relatedXanthomonasspecies, non-Xanthomonasspecies, saprophytic bacteria, and healthyPrunussamples. The efficiency of the developed protocol was evaluated with field samples of 14Prunusspecies and rootstocks. For symptomatic leaf samples, the protocol was very efficient even when washed tissues of the leaves were directly amplified without any previous DNA extraction. For samples of 117 asymptomatic leaves and 285 buds, the protocol was more efficient after a simple DNA extraction, andX. arboricolapv. pruni was detected in 9.4% and 9.1% of the 402 samples analyzed, respectively, demonstrating its frequent epiphytic or endophytic phase. This newly developed real-time PCR protocol can be used as a quantitative assay, offers a reliable and sensitive test forX. arboricolapv. pruni, and is suitable as a screening test for symptomatic as well as asymptomatic plant material.

scholarly journals Detection limit of viruses in mosquitoes by one-step real-time reverse transcription quantitative PCR

2020 ◽  
Author(s):  
Zhaoyang Tang ◽  
Hanano Yamada ◽  
Carina Kraupa ◽  
Sumejja Canic ◽  
Núria Busquets ◽  
...  
Keyword(s):  
Detection Limit ◽  
Quantitative Polymerase Chain Reaction ◽  
Detection Limits ◽  
Mass Rearing ◽  
Qpcr Assay ◽  
Detection Sensitivity ◽  
Infected Mosquito ◽  
Female Population ◽  
Total Rna ◽  
One Step

Abstract BackgroundMosquitoes are the deadliest animals in the world. Their ability to carry and spread diseases to humans causes millions of deaths every year. Due to the lack of efficient vaccines, the control of mosquito-borne diseases often relies on management of the vector. Traditional control methods such as source reduction and chemical insecticides, have proven not to be sufficient to prevent the proliferation and spread of mosquito populations. The sterile insect technique (SIT) is an additional control method that can be combined with other control tactics to suppress specific mosquito populations. The SIT requires the mass-rearing and release of sterile males that would induce sterility in the wild female population. Samples collected from the environment for laboratory colonization, as well as released males, should be free from mosquito-borne viruses (MBV). Therefore, efficient detection methods with defined detection limits for MBV are required. Although a one-step reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) method was developed to detect arboviruses in human and mosquito samples, its detection limit in mosquito samples has yet to be defined. MethodsWe evaluated the detection sensitivity of one step RT-qPCR for targeted arboviruses in large mosquito pools, using pools of non-infected mosquitoes of various sizes (165, 320 and 1600 mosquitoes) containing one infected mosquito body with defined virus titers of chikungunya virus (CHIKV), usutu virus (USUV), West Nile (WNV) virus and Zika virus (ZIKV).ResultsCHIK, USUV, ZIKV, and WNV virus were detected in all tested pools using the RT-qPCR assay. Moreover, in the largest mosquito pools (1600 mosquitoes), RT-qPCR was able to detect the targeted viruses using different total RNA quantities (10, 1, 0.1 ng per reaction) as a template. Correlating the virus titer with the total RNA quantity allowed predicting the maximum number of mosquitoes per pool in which the RT-qPCR can theoretically detect the virus infection. The corresponding equation uses a Ct value of 36 as a cut-off value for virus detection and a virus copy number of 108 for the positive mosquito body. Based on this formula, the detection limits of CHIK, USUV, ZIKV and WNV were 5.08 x 105, 8.74 x 106, 2.33 x 107, and 5.24 x 105, respectively.ConclusionMosquito borne viruses can be reliably detected by RT-qPCR assay in pools of mosquitoes exceeding 1000 specimens. This will represent an important step to expand pathogen-free colonies for mass-rearing sterile males for programmes that have an SIT component.

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