Applications of Digital PCR for Clinical Microbiology

Kuypers, Jane; Jerome, Keith R.

doi:10.1128/jcm.00211-17

Cited by 208 publications

(177 citation statements)

References 43 publications

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“…While RT-dPCR is more sensitive and suitable for detecting low viral loads, its accessibility is limited by the complexity of the system, cost implications and the inability to multiplex target genes of interest. (29) Several automated rapid nucleic acid amplification tests have recently received FDA approval for emergency use. test, run on the Abbott ID NOW device, can provide results within 13 min.…”

Section: Rt-pcr Assays and Their Performancementioning

confidence: 99%

Diagnosis of COVID-19: Considerations, Controversies and Challenges in South Africa

Dheda¹,

Davids²,

Chang³

et al. 2020

WJCM

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Section: Rt-pcr Assays and Their Performancementioning

confidence: 99%

Diagnosis of COVID-19: Considerations, Controversies and Challenges in South Africa

Dheda¹,

Davids²,

Chang³

et al. 2020

WJCM

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“…Digital PCR is the third generation of PCR technology, which is an absolute quantitative technology of nucleic acid molecules [20] . Digital PCR can be divided into 3D PCR and droplet digital PCR (ddPCR) according to the micro-reaction generation mode.…”

Section: Discussionmentioning

confidence: 99%

Using of Digital Droplet PCR in the detection of Mycobacterium tuberculosis DNA with FFPE and Cytological samples

Cao

Wei

et al. 2020

Preprint

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Backgrounds: Accurate diagnosis for TB is essential for TB control. Droplet digital PCR (ddPCR) is a technology that has high sensitivity. However, the use of ddPCR for the detection of TB pathogen in pathological samples is not been fully studied.Methods: A total of 88 samples from the patients who were suspected of tuberculosis were involved in this study, including 65 formalin-fixed and paraffin-embedded (FFPE) specimens and 22 cytological samples. Digital Droplet PCR was used to compare the sensitivity with Real-time PCR. The real-time PCR ct value and ddPCR TB abundant ratio were analyzed by SPSS software.Results: Among the 62 samples that were “possible TB” detected by real-time PCR, 34 samples were ddPCR-positive, 18 samples were ddPCR-negative, and 10 samples were in “gray area” by ddPCR. 26 patients that were ddPCR-positive received anti-tuberculosis therapy and 14 cases of them benefit from the treatment.Conclusions: ddPCR is more sensitive in the detection of TB than Real-time PCR. ddPCR methods can be used as an additional means for the diagnosis of TB from pathological samples.

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“…Unfortunately, our Octaplex assay standardized with the ARMWG material gave significantly higher values for the samples from the CAP/QCMD surveys, leading to a lack of confidence in the quantitative values obtained. In the absence of available WHO standards for each of the known adenovirus groups, droplet digital PCR quantification of adenovirus would probably be the most accurate method to determine the true quantity of samples (39,40). Given the complex nature of the Octaplex assay with the number of cross-reactions seen for some primers, this would probably best be done with individual assays with single-group primer/probe sets.…”

Section: G G a C G C C T C G G A G Ta C C T C A G A A C A A G T Tt A mentioning

confidence: 99%

Comparison of Three Adenovirus Quantitative PCR Assays with ATCC Reference Strains and Clinical Samples

et al. 2019

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Adenoviruses (AdV) have been associated with a variety of human diseases and are recognized as causing significant morbidity and mortality in immunocompromised or transplant patients. Quantification of AdV DNA in plasma is notoriously difficult due to the genetic diversity of the 71 different serotypes identified to date. There is no World Health Organization standard available to harmonize quantitative data, so results between labs vary widely. In this study, we compared a laboratory-developed multiplex PCR assay with primers and probes specific for each group (A to G) and subgroup E4 (Octaplex) to one with a single primer and probe set (modified from N. Jothikumar et al., Appl Environ Microbiol 71:3131–3136, 2005) and one utilizing bisulfite pretreatment of DNA to reduce variation prior to amplification (Genetic Signatures). Our Octaplex assay detected all low-copy-number clinical samples, while the other two assays had subsets of samples that did not amplify. The modified Jothikumar assay failed to efficiently amplify three of the high-copy-number cultured strains, while the Genetic Signatures 3base assay had a positive bias, resulting in higher copies/ml (>0.5 log10) for all culture fluids tested. All three assays resulted in endpoint detection of the available 51 AdV types. Using two different materials to generate a standard curve revealed that the Octaplex TaqMan assay and the modified Jothikumar assay both consistently gave adenovirus levels lower than the commercial platform for AdV culture fluids but not patient samples. This study highlights the differences in detection of AdV between laboratories that can be attributed to both the PCR method, as well as the reference material used for quantitation.

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Applications of Digital PCR for Clinical Microbiology

Cited by 208 publications

References 43 publications

Diagnosis of COVID-19: Considerations, Controversies and Challenges in South Africa

Diagnosis of COVID-19: Considerations, Controversies and Challenges in South Africa

Using of Digital Droplet PCR in the detection of Mycobacterium tuberculosis DNA with FFPE and Cytological samples

Comparison of Three Adenovirus Quantitative PCR Assays with ATCC Reference Strains and Clinical Samples

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