Application of droplet digital PCR to detect the pathogens of infectious diseases

Li, Haiyi; Bai, Ruolan; Zhao, Zhenyu; Tao, Lvyan; Ma, Mingbiao; Ji, Zhenhua; Jian, Miaomiao; Ding, Zhe; Dai, Xiting; Bao, Fukai; Liu, Aihua

doi:10.1042/bsr20181170

Cited by 178 publications

(143 citation statements)

References 45 publications

(51 reference statements)

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“…Once PCR amplification has been carried out within each of these 20 000 droplets, fluorescent positive and negative droplets are measured and the concentration of target DNA is determined by a Poisson algorithm. Although ddPCR has shown to be a technique with potential and has been used for the detection of specific pathogens, its use in the setting of rapid BSI detection has not been explored yet (Yang et al, 2017;Li et al,;Song et al, 2018;. The use of broad-range primers for the amplification of the highly conserved bacterial 16S rRNA and fungal 28S rRNA, and different fluorescence dye-labelled probes, enables the detection of BSIs, discrimination between fungal and bacterial infections, and -in the latter casespecification of their Gram stain differentiation or genus ( Fig.…”

Section: Introductionmentioning

confidence: 99%

Droplet digital polymerase chain reaction for rapid broad‐spectrum detection of bloodstream infections

Wouters

Dalloyaux

Christenhusz

et al. 2019

Microbial Biotechnology

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Summary The droplet digital polymerase chain reaction (ddPCR) is a novel molecular technique that allows rapid quantification of rare target DNA sequences. Aim of this study was to explore the feasibility of the ddPCR technique to detect pathogen DNA in whole blood and to assess the diagnostic accuracy of ddPCR to detect bloodstream infections (BSIs), benchmarked against blood cultures. Broad‐range primers and probes were designed to detect bacterial 16S rRNA (and Gram stain for differentiation) and fungal 28S rRNA. To determine the detection limit of ddPCR, 10‐fold serial dilutions of E. coli and C. albicans were spiked in both PBS and whole blood. The diagnostic accuracy of ddPCR was tested in historically collected frozen blood samples from adult patients suspected of a BSI and compared with blood cultures. Analyses were independently performed by two research analysts. Outcomes included sensitivity and specificity of ddPCR. Within 4 h, blood samples were drawn, and DNA was isolated and analysed. The ddPCR detection limit was approximately 1–2 bacteria or fungi per ddPCR reaction. In total, 45 blood samples were collected from patients, of which 15 (33%) presented with positive blood cultures. The overall sensitivity of ddPCR was 80% (95% CI 52–96) and specificity 87% (95% CI 69–96). In conclusion, the ddPCR technique has considerable potential and is able to detect very low amounts of pathogen DNA in whole blood within 4 h. Currently, ddPCR has a reasonable sensitivity and specificity, but requires further optimization to make it more useful for clinical practice.

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Section: Introductionmentioning

confidence: 99%

Droplet digital polymerase chain reaction for rapid broad‐spectrum detection of bloodstream infections

Wouters

Dalloyaux

Christenhusz

et al. 2019

Microbial Biotechnology

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“…Furthermore, sensitivity, specificity, accuracy, resolution and tolerance is higher (85,86). However, due to the high cost of equipment and tremendous workload, it is challenging to apply digital PCR to the initial stage of epidemic prevention and control, particularly in under-developed areas, on a large scale (87). However, digital PCR will still be extremely useful as it allows for absolute quantification and the detection of complex background samples, it can track the progress of disease and analyzes the viral load.…”

Section: Future Trendsmentioning

confidence: 99%

Molecular diagnosis of COVID‑19: Current situation and trend in China (Review)

Wang²,

Wang

et al. 2020

Exp Ther Med

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COVID-19 is caused by a novel coronavirus (2019-nCoV or SARS-CoV-2) and has become a global public health emergency. Rapid and accurate molecular diagnostic technologies are crucial for the screening, isolation, treatment, prevention and control of COVID-19. Currently, nucleic acid detection-based techniques and rapid diagnostic tests that detect antigens or antibodies specific to 2019-nCoV infections are the primary diagnostic tools. China National Medical Products Administration has opened a special channel for approval of new pharmaceuticals owing to urgent clinical needs, with 18 nucleic acid detection kits, 11 protein detection kits and 1 sequencing-related equipment and supporting software having been approved until April 23, 2020. The current review summarizes the application situation, advantages, disadvantages and associated technology improvement trends of molecular diagnostics for COVID-19 in China, identifies knowledge gaps and indicates future priorities for research in this field. The most effective way to prevent and control COVID-19 is early detection, diagnosis, isolation and treatment. In the clinical application of molecular diagnosis technology, it is necessary to combine pathogenic microbiology, immunology and other associated detection technologies, advocate the combination of multiple technologies, determine how they complement each other, enhance practicability and improve the ability of rapid and accurate diagnosis and differential diagnosis of COVID-19. Contents 1. Introduction 2. Current molecular diagnostic methods of 2019-nCoV, pros and cons and clinical applications 3. Future trends 4. Conclusion

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“…More recently, the advent of droplet digital PCR (ddPCR) has advanced the detection and quantification of parasites, especially parasites with low intensity infections (Hindson et al 2011;Pinheiro et al 2012). It involves absolute endpoint quantification of parasites and is more advantageous over qPCR, as it relies on calibration curves (generated from a sample with known concentration) to give relative quantification (Li et al 2018). This technique has been successfully applied in accurate detection of several viral, bacterial diseases (King et al 2017), and parasitic infections (Wilson et al 2015;Koepfli et al 2016;Ramírez et al 2018).…”

Section: Parasite Detection and Parasite Genetic Diversitymentioning

confidence: 99%

Towards a more healthy conservation paradigm: integrating disease and molecular ecology to aid biological conservation†

Gupta

Robin

Dharmarajan

2020

J Genet

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Parasites, and the diseases they cause, are important from an ecological and evolutionary perspective because they can negatively affect host fitness and can regulate host populations. Consequently, conservation biology has long recognized the vital role that parasites can play in the process of species endangerment and recovery. However, we are only beginning to understand how deeply parasites are embedded in ecological systems, and there is a growing recognition of the important ways in which parasites affect ecosystem structure and function. Thus, there is an urgent need to revisit how parasites are viewed from a conservation perspective and broaden the role that disease ecology plays in conservation-related research and outcomes. This review broadly focusses on the role that disease ecology can play in biological conservation. Our review specifically emphasizes on how the integration of tools and analytical approaches associated with both disease and molecular ecology can be leveraged to aid conservation biology. Our review first concentrates on diseasemediated extinctions and wildlife epidemics. We then focus on elucidating how host-parasite interactions has improved our understanding of the eco-evolutionary dynamics affecting hosts at the individual, population, community and ecosystem scales. We believe that the role of parasites as drivers and indicators of ecosystem health is especially an exciting area of research that has the potential to fundamentally alter our view of parasites and their role in biological conservation. The review concludes with a broad overview of the current and potential applications of modern genomic tools in disease ecology to aid biological conservation.

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Application of droplet digital PCR to detect the pathogens of infectious diseases

Cited by 178 publications

References 45 publications

Droplet digital polymerase chain reaction for rapid broad‐spectrum detection of bloodstream infections

Droplet digital polymerase chain reaction for rapid broad‐spectrum detection of bloodstream infections

Molecular diagnosis of COVID‑19: Current situation and trend in China (Review)

Towards a more healthy conservation paradigm: integrating disease and molecular ecology to aid biological conservation†

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